Legionella Pneumophila: Pathogenesis and Immunity (Infectious Agents and Pathogenesis)

Legionella pneumophila: Pathogenesis and Immunity INFECTIOUS DISEASES AND PATHOGENESIS Series Editors: Mauro Bendinel...

Author: Paul Hoffman | Herman Friedman | Mauro Bendinelli

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Legionella pneumophila: Pathogenesis and Immunity

INFECTIOUS DISEASES AND PATHOGENESIS Series Editors: Mauro Bendinelli, University of Pisa Herman Friedman, University of South Florida College of Medicine Recent volumes in this series: IN VIVO MODELS OF HIV DISEASE AND CONTROL Edited by Herman Friedman, Steven Specter, and Mauro Bendinelli INFECTIOUS DISEASES AND SUBSTANCE ABUSE Edited by Herman Friedman, Catherine Newton, and Thomas W. Klein CHLAMYDIA PNEUMONIAE INFECTION AND DISEASE Infection and Disease Edited by Herman Friedman, Yoshimasa Yamamoto, and Mauro Bendinelli DNA TUMOR VIRUSES Oncogenic Mechanisms Edited by Giuseppe Barbanti-Brodano, Mauro Bendinelli, and Herman Friedman ENTERIC INFECTIONS AND IMMUNITY Edited by Lois J. Paradise, Mauro Bendinelli, and Herman Friedman HELICOBACTER PYLORI INFECTION AND IMMUNITY Edited by Yoshimasa Yamamoto, Herman Friedman, and Paul S. Hoffman HERPESVIRUSES AND IMMUNITY Edited by Peter G. Medveczky, Herman Friedman, and Mauro Bendinelli HUMAN RETROVIRAL INFECTIONS Immunological and Therapeutic Control Edited by Kenneth E. Ugen, Mauro Bendinelli, and Herman Friedman MICROORGANISMS AND AUTOIMMUNE DISEASES Edited by Herman Friedman, Noel R. Rose, and Mauro Bendinelli LEGIONELLA PNEUMOPHILA: PATHOGENESIS AND IMMUNITY Edited by Paul Hoffman, Herman Friedman, and Mauro Bendinelli

A Continuation Order Plan is available for this series. A continuation order will bring delivery of each new volume immediately upon publication. Volumes are billed only upon actual shipment. For further information, please contact the publisher.

Legionella pneumophila: Pathogenesis and Immunity

Paul Hoffman Department of Microbiology Medicine, Division of Infectious Diseases and International Health, UVA Health System, University of Virginia, Charlottesville, VA, USA

Herman Friedman College of Medicine, University of South Florida, Tampa, FL, USA

Mauro Bendinelli University of Pisa, Italy

Paul Hoffman University of Virginia, Charlottesville, VA, USA Herman Friedman University of South Florida, Tampa, FL, USA Mauro Bendinelli University of Pisa, Pisa Italy

ISBN-13: 978-0-387-70895-9

e-ISBN-13: 978-0-387-70896-6

Library of Congress Control Number: 2007926437 © 2008 Springer Science⫹Business Media, LLC All rights reserved. This work may not be translated or copied in whole or in part without the written permission of the publisher (Springer Science⫹Business Media, LLC, 233 Spring Street, New York, NY 10013, USA), except for brief excerpts in connection with reviews or scholarly analysis. Use in connection with any form of information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed is forbidden. The use in this publication of trade names, trademarks, service marks, and similar terms, even if they are not identified as such, is not to be taken as an expression of opinion as to whether or not they are subject to proprietary rights. Printed on acid-free paper. 9 8 7 6 5 4 3 2 1 springer.com

Dedication We dedicate this book to the memory of Dr. Herman Friedman, editor and friend, who passed away on August 25, 2007. As scholar, scientist and teacher, he contributed broadly to the disciplines of medical microbiology and immunology, but he will be best remembered for his books, mentorship of young scientists and for his seminal contributions to our knowledge of the immunobiology of Legionnaires’ disease.

Contents

Preface and Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xiii Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xvii 1. Legionella pneumophila Pathogenesis: Lessons Learned from Genomics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . CHRISTEL CAZALET and CARMEN BUCHRIESER 1. Genomics of Legionella pneumophila . . . . . . . . . . . . . . . . . . . . . . . 1.1. General Features and Organization of the L. pneumophila Genomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Host–Pathogen Interaction—Specific Features of the L. pneumophila Genomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Eukaryotic-Like Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Putative Virulence Factors as Deduced Prom Sequence Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Secretion Machineries—Presence of Particular Many and a Wide Variety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Metabolism as Deduced from the Genome Sequence . . . . . . . . . . . . 4. Clues to Adaptation—Regulation and Signal Transduction . . . . . . . 5. Comparative Genomics of Legionella pneumophila . . . . . . . . . . . . . 5.1. The Specific Gene Complements of L. pneumophila Paris, Lens and Philadelphia . . . . . . . . . . . . 5.2. Genomic and Pathogenicity Islands . . . . . . . . . . . . . . . . . . . . . 5.3. Plasmids of Legionella . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.4. Variability at the Gene Level . . . . . . . . . . . . . . . . . . . . . . . . . . . 6. Comparative Genomics— Legionella, Coxiella and Other Intracellular Pathogens . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1. Genome Comparison of Legionella pneumophila and Coxiella burnetii . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2. Genome Redundancy—Gene Family Expansion in Legionella . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7. Comparative Genomics of Legionella pneumophila and the Genus Legionella —a Perspective . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1 1 1 3 3 11 13 14 15 17 17 18 20 22 23 23 24 25 26 vii

viii

Contents

2. Iron Assimilation and Type II Protein Secretion . . . . . . . . . . . . . . . . . . NICHOLAS P. CIANCIOTTO

33

1. Iron Assimilation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1. The Importance of Iron for Legionella pneumophila . . . . . . . . 1.2. Legionella Siderophores . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.3. Ferrous Iron Transport . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.4. The L. pneumophila Iron Acquisition/Assimilation Locus . . . . 1.5. Cytochrome C Maturation and Iron Assimilation . . . . . . . . . . . 1.6. Legionella Ferric Reductases . . . . . . . . . . . . . . . . . . . . . . . . . . 1.7. Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Type II Protein Secretion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Type II Protein Secretion in Gram-Negative Bacteria . . . . . . . 2.2. Discovery of Type II Secretion in Legionella pneumophila . . . 2.3. Factors Secreted by the Type II System of L. pneumophila . . . 2.4. Role of Type II Secretion in Pathogenesis . . . . . . . . . . . . . . . . 2.5. Role of Type II Secretion in Environmental Survival . . . . . . . . 2.6. Concluding Comments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

33 33 34 37 38 38 39 39 40 40 41 42 43 44 45 45

3. The Dot/Icm Type IVB Secretion System . . . . . . . . . . . . . . . . . . . . . . . JASON J. LEBLANC and JOSEPH P. VOGEL

49

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Dot/Icm Type IV Secretion System . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Structural Components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Adaptor/Chaperone Complexes . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Regulation of Effector Export . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Genetic Regulation of the Dot/Icm System . . . . . . . . . . . . . . . . . . . 4. Possible Effector Proteins and Associated Functions . . . . . . . . . . . . 4.1. RalF . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. The Sid Family . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3. LidA and SidM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4. LepA and LepB . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.5. Ylf and the Vips . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Temporal Modulation of the LCV Surface . . . . . . . . . . . . . . . . . . . . 6. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

49 50 52 53 54 54 55 55 56 56 57 57 58 59 59

4. Life Cycle, Growth Cycles and Developmental Cycle of Legionella pneumophila . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . RAFAEL A. GARDUÑO

65

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. The Life Cycle of L. pneumophila as Currently Understood . . . . . . 2.1. Attachment and Invasion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

65 66 67

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2.2. Post-Internalization Events and Intracellular Growth . . . . . . . . 2.3. End of Replication and Exit . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4. What Role Do Ciliates Play in the Life Cycle of L. pneumophila? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. The Growth Cycles of L. pneumophila . . . . . . . . . . . . . . . . . . . . . . . 4. The Developmental Cycle of L. pneumophila . . . . . . . . . . . . . . . . . 4.1. Legionella pneumophila Development Along the Extracellular Growth Cycle . . . . . . . . . . . . . . . . . . . 4.2. MIF: The Result of L. pneumophila Intracellular Differentiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3. The Central Role of MIFs in the Study of L. pneumophila Virulence . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

68 69 69 70 72 73 76 77 78 79

5. Legionella in the Environment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . BARRY S. FIELDS

85

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. The Distribution of Legionella as a Function of Various Detection Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Natural Environments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Man-Made Environments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Interaction with Other Organisms . . . . . . . . . . . . . . . . . . . . . . . . . . . 6. Association with Biofilms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

85

6. Regulation of the Legionella pneumophila Life Cycle . . . . . . . . . . . . . RACHEL L. EDWARDS and MICHELE S. SWANSON

95

1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Life Cycle of L. pneumophila . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Broth Model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Amino Acid Availability Governs Differentiation . . . . . . . . . . . . . . 4.1. Pht Family of Transporters . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Stringent Response . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Evidence that Other Factors Trigger Differentiation . . . . . . . . . . . . 5.1. Acetyl-phosphate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6. Transcriptional Control of Differentiation via Sigma Factors . . . . . 6.1. RpoS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2. RpoN and FleQ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.3. FliA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7. Post-Transcriptional Control of Differentiation . . . . . . . . . . . . . . . . 7.1. LetA/LetS Two-Component System . . . . . . . . . . . . . . . . . . . . . 7.2. Carbon Storage Regulatory (Csr) System . . . . . . . . . . . . . . . . . 7.3. Hfq and Small RNAs (sRNAs) . . . . . . . . . . . . . . . . . . . . . . . . .

95 95 97 98 98 100 101 101 102 103 103 103 104 104 105 106

85 87 88 90 90 91

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Contents

8. Regulation of the Dot/Icm Type IV Secretion System . . . . . . . . . . . 107 9. Genomic Methods of Studying Differentiation . . . . . . . . . . . . . . . . . 108 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108 7. Microbial Physiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113 PAUL S. HOFFMAN 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Intracellular Growth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Morphology, Ultrastructure, and Surface Proteins . . . . . . . . . . . . . 3.1. Envelope Structure and Lipopolysaccharide (LPS) . . . . . . . . 3.2. Outer Membrane Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Secreted Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4. Motility and Chemotaxis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Respiratory Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Central Intermediary Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . 6. Biosynthetic Capacity and Amino Acid Auxotrophies . . . . . . . . . . 7. Protection from Oxidative Stress . . . . . . . . . . . . . . . . . . . . . . . . . . 8. Heat Shock Response . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9. Stationary Phase Genes and Cell Wall Assembly . . . . . . . . . . . . . . 10. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

113 114 115 116 116 117 118 119 119 123 124 126 127 128 128

8. Legionnaires’ Disease—Clinical Picture . . . . . . . . . . . . . . . . . . . . . . . . 133 THOMAS J. MARRIE 1. 2. 3. 4.

5. 6. 7. 8.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Epidemiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Clinical Features of Legionnaires’ Disease . . . . . . . . . . . . . . . . . . . . Diagnosis of Legionella Infection . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Routine Laboratory Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Specific Tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pontiac Fever . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Nosocomial Legionnaire’s Disease . . . . . . . . . . . . . . . . . . . . . . . . . . Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

133 133 137 140 140 140 141 143 143 144 144

9. Legionella pneumophila: Innate and Adaptive Immunity . . . . . . . . . . . 151 HERMAN FRIEDMAN, CATHERINE NEWTON and THOMAS KLEIN 1. 2. 3. 4.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Legionella Immunogens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Innate Immunity to L. pneumophila . . . . . . . . . . . . . . . . . . . . . . . . . Adaptive Immunity to L. pneumophila Infection . . . . . . . . . . . . . . .

151 152 153 156

Contents

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5. Immune Mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6. Discussion and Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

157 164 166 167

10. Nonspecific Stimulation of Immunity Against Legionella . . . . . . . . . . 173 JAMES ROGERS, AMAL HAKKI and HERMAN FRIEDMAN 1. 2. 3. 4. 5. 6. 7. 8. 9.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Stimulation of Immunity by Natural Products . . . . . . . . . . . . . . . . . Antibacterial and Antiviral Effects of Epigallocatechin Gallate . . . . Modulation of Cytokines by Natural Products . . . . . . . . . . . . . . . . . The Effects of EGCG on Cytokine Production by Legionella-Infected Dendritic Cells . . . . . . . . . . . . . . . . . . . . . . . The Effects of EGCG on Co-stimulatory Molecule Expression by Legionella-infected Dendritic Cells . . . . . . . . . . . . . The Effects of EGCG on Chemokine Production by Legionella-infected Dendritic Cells . . . . . . . . . . . . . . . . . . . . . . . Immunomodulatory Effects of Natural Products on TLR Expression and Signal Transduction . . . . . . . . . . . . . . . . . . Summary and Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

173 174 174 175 176 177 178 179 181 182

11. Interaction of Legionella pneumophila with Amoeba . . . . . . . . . . . . . . 185 MAËLLE MOLMERET, MARINA SANTIC, and YOUSEF ABU KWAIK 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Legionellae: A Facultative Intracellular Pathogen of Free-Living Amoebae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. The Role of Amoebae in Persistence of Legionella in the Environment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. The Role of Amoebae in Pathogenesis of Legionella . . . . . . . . . . . . 5. Entry of L. pneumophila into Protozoa . . . . . . . . . . . . . . . . . . . . . . . 6. Intracellular Trafficking and Multiplication of L. pneumophila . . . . 7. Role of the dot/icm Genes in Evasion of the Endocytic Pathway . . . 8. Egress of L. pneumophila from Amoebae . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

185 187 188 189 190 191 192 194 196

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 203

Preface and Introduction

The summer of 2006 marked the 30th anniversary of the outbreak of Legionnaires’ disease, an acute pneumonia that occurred among veterans who had gathered in Philadelphia, Pennsylvania, in 1976 to celebrate the bicentennial of the founding of the United States. Ironically, the nation and world were braced for an epidemic of swine influenza, which never materialized and no one expected the emergence of a new bacterial pathogen, since most in the medical field sincerely believed that all the pathogens of humans were now known. This new bacterial agent of Legionnaires’ disease, Legionella pneumophila (named in memory of the deceased veterans) was a harbinger of diseases to come (emerging pathogens) that has included HIV/AIDS, SARS, Lyme disease, hamburger disease, and many others. The further discovery that L. pneumophila was ubiquitous to aquatic environments worldwide and resided as an intracellular parasite of amoeba and protozoa provided a link between natural environments and human disease. Thus, environmental monitoring, especially of potable water, cooling towers, and related sources, has become a major focus in efforts to control the spread of this disease. Fortunately, as noted in the 1976 epidemic and holds true today, the disease is not spread from human to human. The remarkable ability of L. pneumophila to multiply in alveolar macrophages, as if they were amoebae, has further contributed to our understanding of the disease and stimulated much research in the area of cellular microbiology of pathogenesis. Today, Legionnaires’ disease is both sporadic (community acquired) and epidemic (explosive point sources such as cooling towers) and is on the rise worldwide – attributed to a greater dependence on technology and air conditioning together with an increasingly susceptible population due to medical technology. Despite all the advances in medicine, Legionnaires’ disease continues to have a high mortality rate, not uncommon when pneumonia is involved. Advances in understanding pathogenesis and immunity have benefited greatly from the availability of three genome sequences for L. pneumophila and one for close relative Coxiella burnetii. Both Legionella and Coxiella produce survival forms and display a developmental cycle which contribute to pathogenesis, persistence, and resistance to biocides in natural environments. There have been many major developments in the Legionella field ranging from strategies to treat infections to how xiii

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this organism interacts with innate immune mechanisms to promote infections. This book represents the first collection of chapters written by experts in each of the specialty areas in this field. Unlike past books, which have been proceedings from international meetings, we have provided an opportunity for each of the authors to present “state of the art” detailed descriptions of specific areas that permit interesting reading for the non-expert and a solid reference for those researchers active in this field. The authors of each of the chapters in this book are internationally recognized experts concerning Legionella infections. The first chapter by Drs. Christel Cazalet and Carmen Buchreiser from the Institute Pasteur, Paris, France, concerns the molecular biology and genetics of L. pneumophila pathogenesis. The authors describe in-depth laboratory investigations of the genetic differences between the numerous strains of these opportunistic bacteria and provide important new information how these microbes interact with environmental factors and their host. The next chapter by Dr. Nicholas Cianciotto, Northwestern University Medical School, Chicago, Illinois, stresses important physiological aspects of Legionella–host interactions, especially the important role of iron assimilation and the nature and mechanism of protein secretion by these bacteria. Drs. J. Le Blanc, Dalhouse University, Halifax, Nova Scotia from Canada, and Joseph P. Vogel from St. Louis, Missouri, then describe the metabolic activity of these ubiquitous microbes, especially the nature and mechanism of their secretion system. Dr. Rafael Garduño, Dalhousie University, Halifax, Nova Scotia Canada, then discusses pathogenesis and immunity to that bacterium, including some unique features of the life as well as developmental cycle of L. pneumophila in a host, both human and protozoa in their natural habitat. Dr. Barry Fields, Centers for Disease Control and Prevention, Atlanta, Georgia, then presents detailed observations how Legionella are widely dispersed in the environment, especially freshwater habitats. Drs. Rachel Edwards and Michele Swanson, University of Michigan, Ann Arbor, Michigan, describe how the life cycle of these bacteria is regulated and information necessary to control the presence of the bacteria, especially in the environment, and treatment modalities with antibiotics which abrogate bacterial replication and/or disease progression. Although no effective vaccine has yet been developed, information concerning interaction of these Legionella with the human host and how they infect host cells, as well as various antigenic structures of the bacteria important for infectivity and replication, provide a foundation necessary for developing protective vaccines which can induce adaptive immunity. Dr. Paul Hoffman, University of Virginia, provides new information about the interesting unique physiology of L. pneumophila and other species of these bacteria. He compares different aspects of their physiology in the human host vs. replication in amoeba and protozoa. Dr. Thomas J. Marrie, Walter C. Mackenzie Health Science Center, Edmonton, Alberta, Canada, then describes medical and clinical aspects of Legionnaire’s disease which first attracted attention in the epidemic pneumonia outbreak in a hotel in Philadelphia during the annual convention of Pennsylvania State Legionnaires after which the disease is named. His chapter describes clinical aspects of the


xv

infection in individuals showing respiratory symptoms. It is also now widely recognized that many individuals have subclinical exposure to Legionella, as evident by high titers of specific humoral antibody and marked in vitro blastogenic responsiveness of their blood lymphocytes to Legionella antigen. Drs. H. Friedman, C. Newton, and Thomas W. Klein, University of South Florida, discuss in detail the exploding new knowledge concerning immunity to L. pneumophila in terms of newer information about innate and adaptive specific immune responses to these ubiquitous opportunistic intracellular bacteria which primarily cause serious infection in immunocompromised individuals but which have their natural habitat single cell protozoa. They apparently interact with human hosts because of their ubiquitous presence in warm lakes, airconditioning cooling towers, circulating warm water in plumbing, and drinking water in institutions such as hospitals. The next chapter by Drs. J. Rogers, A. Hakki, and H. Friedman, also from the University of South Florida, is concerned with information how nonspecific immune stimulators modify host susceptibility to infection by opportunistic bacteria such as Legionella. This chapter reviews nonspecific modulators of both innate and adaptive immune mechanisms which impact the host immune response to an opportunistic pathogen like Legionella. Plant-derived nonspecific immunoenhancing substances are being investigated worldwide and various small molecular weight substances used for centuries as “folk medicine” to ameliorate infections have been shown clinically to significantly enhance host resistance to ubiquitous microbial infections. Drs. Maelle Molmeret and Yousef Abu Kwaik, University of Louisville, Louisville, Kentucky, then describe in detail information concerning L. pneumophila interactions with amoeba in vitro. Knowledge about such interactions is important to understand the nature and mechanism whereby these bacteria interact with phagocytic cells of the human host and the many biochemical and physiological features of legionellae common to their uptake and regulation in different cell types. The editors as well as authors of individual chapters believe the many ongoing studies of host immunity to ubiquitous opportunistic pathogens like L. pneumophila will undoubtedly lead to more effective control methods to prevent or ameliorate infections by such microorganisms, which still account for many thousands of infections annually in the United States alone. Many details concerning the nature and mechanism of interactions between host and opportunistic intracellular organisms like Legionella, which preferentially infect phagocytic cells, are being clarified and provide important knowledge directly related to mechanisms of host resistance to other important intracellular opportunistic pathogens, including Mycobacteria which cause tuberculosis. We thank Ms. Ilona Friedman for continuing excellent editorial assistance correlating and assisting in preparation of manuscripts for this book in the series. Paul S. Hoffman Herman Friedman Thomas W. Klein

Contributors

Carmen Buchrieser • Unité de Génomique des Microorganismes Pathogegens, Institut Pasteur, Paris, FRANCE Christel Cazalet • Unité de Génomique des Microorganismes Pathogegens, Institut Pasteur, Paris, FRANCE Nicholas P. Cianciotto • Department of Microbiology-Immunology, Northwestern University Medical School, Chicago, IL Rachel L. Edwards • Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, MI Barry S. Fields • Centers for Disease Control and Prevention, Atlanta, GA Herman Friedman • Department of Molecular Medicine, University of South Florida, College of Medicine, Tampa, FL Rafael A. Garduño • Department of Microbiology and Immunology and Department of Medicine, Dalhousie University, Halifax, Nova Scotia, CANADA Amal Hakki • Department of Molecular Medicine, University of South Florida College of Medicine, Tampa, FL Paul S. Hoffman • Division of Infectious Diseases and International Health, Department of Medicine, University of Virginia Health Systems, Charlottesville, VA Thomas W. Klein • Department of Molecular Medicine, University of South Florida College of Medicine, Tampa, FL Yousef Abu Kwaik • Department of Microbiology and Immunology, University of Louisville, Louisville, KY Jason J. LeBlanc • Department of Microbiology and Immunology, Dalhousie University, Halifax, Nova Scotia, CANADA Thomas J. Marrie • Department of Medicine, University of Alberta, Edmonton, Alberta, CANADA

xvii

xviii

Contributors

Maelle Molmeret • Department of Microbiology and Immunology, University of Louisville, Louisville, KY Catherine Newton • Department of Molecular Medicine, University of South Florida College of Medicine, Tampa, FL James Rogers • Department of Molecular Medicine, University of South Florida College of Medicine, Tampa, FL Marina Santic • Department of Microbiology and Parasitology, University of Rijeka, Rijeka, CROATIA Michele S. Swanson • Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, MI Joseph P. Vogel • Department of Molecular Microbiology, Washington University, St. Louis, MO

1 Legionella pneumophila Pathogenesis: Lessons Learned from Genomics Christel Cazalet and Carmen Buchrieser

The last years have seen a giant step in the genomics of Legionella pneumophila. The establishment and publication of the complete genome sequences of three clinical L. pneumophila isolates in 2004 paves now the way for major breakthroughs in understanding the biology of L. pneumophila in particular and Legionella in general (Cazalet et al. 2004; Chien et al. 2004). This chapter starts with a description of the three genome sequences of L. pneumophila strain Paris, L. pneumophila strain Lens and L. pneumophila strain Philadelphia 1 and then highlights the characteristic features and common traits of the Legionella genomes. Emphasis is given on putative virulence and Legionella life cycle–related functions. In the second part, we focus on the comparison of these three complete genome sequences, in order to learn about the plasticity of the Legionella genomes and the possible mechanisms involved. In the third part we compare the Legionella genomes with those of their closest relative, Coxiella burnetii, to try to trace the genetic basis of their common features. Finally, other Legionella species, for which genomic data are available, are discussed briefly and future perspectives in Legionella genomics are presented.

1. Genomics of Legionella pneumophila 1.1. General Features and Organization of the L. pneumophila Genomes The complete genome sequences of three strains of L. pneumophila (Paris, Lens and Philadelphia 1) are available at the time of writing (Cazalet et al. 2004; Chien et al. 2004). Legionella pneumophila has a single, circular chromosome, 3,503 610 base pairs (bp) (Paris), 3,345 687 bp (Lens) and 3,397 754 bp (Philadelphia 1) in size, with an average G ⫹ C content of 38% (Table 1.1; Figure 1.1. Strains Paris and Lens each contain a plasmid, 131.9 kb and 59.8 kb in size, respectively. In strain Philadelphia 1 no plasmid was identified. The genomes contain each ⬃3000 genes distributed fairly evenly between the two strands (⬃57% on the leading strand) and accounting for ⬃88% of the potential coding capacity. No function 1

2

Christel Cazalet and Carmen Buchrieser

TABLE 1.1. General features of the three sequenced Legionella pneumophila genomes.

Size of the chromosome (bp) G ⫹ C content Total number of protein-coding genes Average length of protein-coding genes Number of rRNA operons (16S-23S-5S) Number of tRNA genes Percentage coding Plasmid1 Number of strain specific genes Number of orthologous genes

L. pneumophila Paris

L. pneumophila Lens

L. pneumophila Philadelphia

3,503,610

3,345,687

3,397,754

38.3% (37.4%) 3077 (141)

38.4% (38.4%) 2931 (57)

38% 2953

331*

333*

338*

3

3

3

43 87.9% (92%) 1 (131.9 kb) 313 (125)

43 88% (83.7%) 1 (59.8 kb) 259 (44)

43 89.8 – 335

2500

2500

2500

*codons 1kilobase-pairs

() plasmid related

could be predicted for 42.1% of the L. pneumophila Paris, 44.1% of the L. pneumophila Lens and for 28% of the L. pneumophila Philadelphia 1 genes. A high proportion of the predicted genes (21% for strain Paris, 20.4% for strain Lens, 17% for strain Philadelphia 1) are unique to the genus Legionella. Comparative analysis of the genome structure of the three L. pneumophila genomes showed high colinearity, with only few translocations, duplications, deletions or inversions (Figure 1.2). Principally, the genomes contain three large plasticity zones, where the synteny is disrupted: a 260kb inversion in strain Lens with respect to strains Paris and Philadelphia 1, a 130kb fragment which is partly similar partly different and which is inserted in a different genomic location in strains Paris and Philadelphia 1 but which is truncated in strain Lens and the chromosomal region carrying the Lvh type IV secretion system (Figure 1.2). The Lvh type IV secretion system, previously described in strain Philadelphia (Segal et al. 1999) is encoded by a 36 kb region (strain Paris) or a 45 kb region (strain Philadelphia 1) that can be either carried on a multi-copy plasmid or be integrated into the chromosome. Further diversity on the genome level is present as deletions and insertions of several smaller regions were identified in each strain, as well as regions where the gene content is variable. Finally, also variations at the gene level are present as discussed later. The sequences and their analysis are accessible at http://genolist.pasteur.fr/LegioList; http://genome3.cpmc.columbia.edu/⬃legion/project/.

1. Legionella pneumophila Pathogenesis

3

0 10

3.0

8

1

9

0.5 2

Legionella pneumophila strain Paris

3

3 503 610 bp

1.0

2.5 4

7 2.0

65

1.5

FIGURE 1.1. Circular genome map of L. pneumophila strain Paris and the specific genes with respect to strain Lens and Philadelphia. From the outside—circle 1: Strain Paris genes on the ⫹ and – strands, respectively. Red line, inversion in strain Lens. Numbers indicate their position: 1 lvh-lvr type IV secretion system (lvrABC, lvhB2B3B4B5, lvrD, lvhB6B8B9B10B11D4, lvrE); 2 dot/icm type IV secretion system (icmTSRQOMLKEGCDJBF); 3 mip, 4 lspA, 5 lspDE, 6 htrA, 7 lspFGHIJK, 8 enhABC, 9 dot/icm type IV secretion system (icmVWX and dotABCD), 10 momp; circle 2: specific genes of strain Paris with respect to strain Lens; circle 3: specific genes of strain Paris with respect to strain Philadelphia; circle 4: G/C bias (G ⫹ C/G-C) of strain Paris; circle 5: G ⫹ C content of strain Paris with ⬍32.5% G ⫹ C short length, between 32.5% and 44.1% medium length and with ⬎44.1% G ⫹ C long lines. The scale (Mb) is indicated on the outside, origin of replication at position 0.

2. Host–Pathogen Interaction—Specific Features of the L. pneumophila Genomes 2.1. Eukaryotic-Like Proteins Protozoa are essential for the growth of Legionella. The interaction of L. pneumophila with aquatic protozoa seems to have generated a pool of virulence traits during evolution, which allow Legionella to infect also human cells. This is reflected in the genome sequence of this pathogen, as it codes an unexpected

Christel Cazalet and Carmen Buchrieser A

B

L. pneumophila

Lens

2

1

2

L. pneumophil a Philadelphia

4

2 4

3 2

3

L. pneumophila

Paris

L. pneumophila

Paris

FIGURE 1.2: Synteny plot of the chromosomes of strains Paris and Lens (A) and strains Paris and Philadelphia (B). Inversions between the genomic sequences are represented in blue. Genome-wide synteny is disrupted by a 260 kb inversion 1 and a 130 kb plasticity zone in strain Paris 2 as well as by some smaller clusters. The insertion 2 is also present in strain Philadelphia but in a different chromosomal location as well as the lvh secretion system 3. Synteny is also disrupted by an insertion of a 65 kb pathogenicity island in strain Philadelphia 4. Plots were created with the mummer software package developed by Tigr (http://www.tigr.org).

high number and variety of eukaryotic-like proteins, which are probably able to interfere in all different steps of the infectious cycle by mimicking functions of eukaryotic proteins. The large number and wide variety of eukaryotic-like proteins present in the L. pneumophila genomes is to date unique for a prokaryotic genome. Half of these proteins show the strongest similarity to eukaryotic proteins and the others contain motifs known to be implicated in protein–protein interactions mostly or uniquely present in eukaryotes, suggesting that L. pneumophila may have developed specific mechanisms to cross-talk with its eukaryotic hosts. It is thus likely that these proteins confer to L. pneumophila the capacity to subvert host functions and to modulate them to its advantage. When comparing the three sequenced genomes, it becomes evident that most of these proteins should fulfill important functions, as nearly all are conserved in the three sequenced genomes (Tables 1.2A and 1.2B). Recently, analysis of the gene content of 180 strains by DNA–DNA hybridization showed that the eukaryotic-like genes identified during sequence analysis are with small exceptions, conserved in all the strain tested (Cazalet et al. 2005a). These results support the hypothesis that they are required for intracellular growth and that they may play a role in virulence. Some of these proteins are predicted to have a role in different regulatory pathways, others may be secreted into the eukaryotic cell and thus be implicated

34 39

lpp0321

lpp1157

RNA binding protein precursor pyruvate decarboxylase

38 36 39

36 41 39 39 36 39 40

lpp2832

plpp0050

lpp0634

lpp0965

lpp2748 lpp2128

lpp0489 lpp0955 lpp0578 lpp0379 lpp1033

DegP protease

phytanoyl-coA dioxygenase sphingosine-1-phosphate lyase glucoamylase cytokinin oxidase phytanoyl coA dioxygenase Hypothetical protein ectonucleoside triphosphate diphosphohydrolase (apyrase)

39

38

lpp1522

thiamine biosynthesis protein NMT-1 nuoE NADH dehydrogenase I chain E hypersensitive induced response protein hypothetical protein

39

lpp0702

exoA exodeoxyribonuclease III

G–C(%) 38

L.p. Paris lpp1647

Predicted product purC

lpl0465 lpl0925 lpl0554 lpl0354 lpl1000

lpl2621 lpl2102

lpl0935

lpl0618

–

lpl2701

lpl1461

lpl1162

–

lpl0684

L.p.Lens lpl1640

39 39 37 40 39

36 41

40

39

–

38

39

39

–

39

G–C(%) 39

lpg0422 lpg0894 lpg0515 lpg0301 lpg0971

lpg2694 lpg2176

lpg0903

lpg0584

–

lpg2785

lpg1565

lpg1155

lpg0251

lpg0648

L.p.Phila lpg1675

39 40 37 40 40

36 40

40

39

–

37

40

40

37

39

G–C(%) 40

(Continued)

(P42042 Arxula adeninivorans) (NP_484368.1 Nostoc sp.) (EAA70100.1 Gibberella zeae) (CAD21525.1 Taenia solium) nucleoside phosphatase signature conserved (Q9MYU4 Sus scrofa)

(AAN17462.1 Hordeum vulgare subsp. Vulgare) (XP_306643.1 Anopheles gambiae) (NP_189431.2 Arabidopsis thaliana) (XP_372144.1 Homo sapiens) (NP_775139.1 Rattus norvegicus)

Best Hit BLASTp (AAR06292.1 Nicotania tabacum) (EAA20230.1 Plasmodium yoelii yoelii) (AAL07519 Solanum tubeosum) (AAB16855.1 Arabidopsis thaliana) (AAC64375.1 Botryotinia fuckeliana) (BAA25988.1 Homo sapiens)

TABLE 1.2A. Proteins with the highest similarity score to eukaryotic proteins and their distribution in the three sequenced strains.

lpp1127

lpp1167 lpp2626 lpp1439

uridine kinase ser/thr proteine kinase domain serine threonine protein kinase

36 41

lpp1665 lpp1959

Ca2 ⫹ -transporting ATPase

39 34

lpp2468 lpp1824

33 32 36

37

38

35

lpp2747

lpp0358

39

lpp1880

hypothetical protein

35

lpp2134

38

lpp3071

SAM dependent methyltransferase ectonucleoside triphosphate diphosphohydrolase (apyrase) SAM dependent methyltransferase cytochrome P450 nuclear membrane binding protein uracyl DNA glycosylase chromosome condensation 1-like

34

lpp2923

6-pyruvoyl-tetrahydropterin synthase zinc metalloproteinase

G–C(%)

L.p. Paris

Predicted product

TABLE 1.2A. (Continued)

lpl1173 lpl2481 lpl1545

lpl1131

lpl0334

lpl1659 lpl1953

lpl2326 –

lpl2620

lpl1869

lpl2109

lpl2927

lpl2777

L.p.Lens

34 32 35

37

38

36 38

39 –

35

39

36

38

35

G–C(%)

lpg1165 lpg2556 lpg1483

lpg1126

lpg0282

lpg1700 lpg1976

lpg2403 –

lpg2693

lpg1905

lpg2182

lpg2999

lpg2865

L.p.Phila

34 32 36

38

39

37 43

38 –

36

40

36

38

35

G–C(%)

Best Hit BLASTp

(EAA36774.1 Giardia lamblia) Pattern of chromosome condensation regulator conserved (EAA20288.1 Plasmodium yoelii yoelii) (AAB81284.1 Paramecium tetraurelia) (AAM09314.2 Mus musculus) conserved domain conserved domain

(CAE70887.1 Caenorhabditis briggsae) (EAA20288.1 Plasmodium yoelii yoelii) (NP_487786.1 Nostoc sp.) (NP_082559.1 Mus musculus)

(NP_703938.1 Plasmodium falciparum) (AAF56122.1 Drosophila melanogaster) (BAC98835.1 Bombyx mori)

L.p. Paris enhC (lpp2692) lidL(lpp1174)—EnhC paralog lpp1310—EnhC paralog lpp2174—EnhC paralog – ralF (lpp1932) lpp0267 lpp2626 lpp1439 lpp2065 lpp0037 plpp0098 lpp2058 lpp0750 lpp2061 lpp2270 lpp0503 lpp1905 lpp1683 lpp2248 lpp0202 lpp0469 lpp2517 lpp1100 lpp0126 lpp0356 lpp2522 lpp0547

41 40 45 34 38 32 36 37 38 37 38 35 39 34 38 35 33 39 38 38 36 48 39 38 39 40

G–C% 39 38

L. p.Lens enhC (lpl2564) lidL (lpl1180)—EnhC paralog lpl1307—EnhC paralog lpl1303—EnhC paralog lpl1059—EnhC paralog ralF lpl1919 lpl0262 lpl2481 lpl1545 lp2055 lpl0038 – lpl2048 lpl0732 lpl2051 lpl2242 lpl0479 – lpl1682 lpl2219 – lpl0445 lpl2370 – lpl0111 – lpl2375 lpl0523 41 39 45 34 39 32 35 37 39 – 38 35 39 34 36 – 34 39 – 38 37 – 39 – 39 41

G–C(%) 39 39

L.p.Philadelphia enhC (lpg2639) lidL (lpg1172)—EnhC paralog lpg1356—EnhC paralog lpg2222—EnhC paralog lpl1062—EnhC paralog ralF (lpp1950) lpg0208 lpg2556 lpg1483 lpg2214 lpg0038 – – lpg0695 – lpg2322 lpg0436 – lpg1718 lpg2300 – lpg0403 lpg2452 – lpg0112 – lpg2456 lpg0483 41 41 44 35 38 32 36 42 38 – – 36 – 35 37 – 34 39 – 39 37 – 39 – 40 42

–C(%) 39 39

(Continued)

4 sel-1 domains 3 sel-1 domains 7 sel-1 domains sec7 domain ser/thr kinase domain ser/thr kinase domain ser/thr kinase domain ankyrin repeat ankyrin repeat ankyrin repeat ankyrin repeat ankyrin repeat ankyrin repeat ankyrin repeat ankyrin repeat ankyrin repeat ankyrin repeat ⫹ SET ankyrin repeat ankyrin repeat ankyrin repeat ankyrin repeat ankyrin repeat ankyrin repeat ankyrin repeat ankyrin repeat ankyrin repeat

Motif identified 21 sel-1 domains 6 sel-1 domains

TABLE 1.2B. Legionella pneumophila proteins-encoding domains preferentially found within eukaryotic proteins and their distribution in the three sequenced strains.

G–C% 34 35 40 38 39 36

34

43 39 35

L.p. Paris – – – – – lpp2082

lpp2486

– lpp0233 lpp2887

TABLE 1.2B. (Continued)

– lpl0234 –

–

L. p.Lens lpl1681 lpl2344 lpl2058 – – lpl2072

– 39 –

–

G–C(%) 34 35 40 – – 36

lpg2224 lpg0171 lpg2830

–

L.p.Philadelphia – – lpg2128 lpg0402 lpg2131 lpg2144

43 40 35

–

–C(%) – – 37 38 39 37

Motif identified ankyrin repeat ankyrin repeat ankyrin repeat ankyrin repeat ankyrin repeat F-box domain ⫹ ankyrin repeat F-box domain ⫹ coiled-coil F-box domain F-box domain 2 U-box domains


9

in different stages of the intracellular cycle of Legionella like in invasion, trafficking into host cell, modulation of host-cell functions and in evasion. Proteinmotifs predominantly found in eukaryotes, which were identified in the L. pneumophila genomes, are ankyrin, Sel-1 (TPR), U-box and F-box motifs. In Legionella ankyrin repeat proteins might play an important role in the intracellular life cycle as they form the most prominent family of proteins with eukaryotic-like domains. It is also the most heterogeneous protein family of the eukaryotic like proteins, as only twelve ankyrin repeat proteins are shared by all three isolates. In addition, strain Paris, Lens and Philadelphia 1 encode each six, four or two specific ankyrin proteins, respectively (Table 1.2B). Large families of ankyrin repeat proteins are so far only identified in C. burnetii (Seshadri et al. 2003) and Wolbachia pipitentis (Wu et al. 2004) and recently in Rickettsia felis (Ogata et al. 2005), all intracellular bacteria. Ankyrin proteins are multifunctional and involved in many cellular pathways, thus the prediction of the function of the Legionella ankyrin proteins is difficult. They might be involved in the interaction with the host cytoskeleton (Batrukova et al. 2000) or in targeting proteins to the plasma membrane or to the endoplasmic reticulum (HryniewiczJankowska et al. 2002). These protein motifs are frequently found in eukaryotic proteins, but their distribution is rather limited in viruses and bacteria, in both of which they appear to be linked with pathogenicity. Another interesting finding is the presence of F-box and U-box domain containing proteins in the Legionella genomes. One could speculate that these proteins may allow L. pneumophila to interfere with the ubiquitin machinery of eukaryotic cells. Three F-box proteins, one of which is specific for each strain, are present in strains Paris and Philadelphia 1, strain Lens contains two F-box proteins (Table 1.2B). Strain Philadelphia 1 and Paris encode the only U-box protein identified in a prokaryotic genome to date. Ubiquitination is a protein modification generally used by cells to tag proteins that are destined for proteasomal degradation. Ubiquitin is a highly conserved 76 amino acid polypeptide that can be covalently attached to a lysine residue of the target protein by an E3-ubiquitin ligase, which is primarily responsible for providing substrate specificity to ubiquitin conjugation. Several classes of ubiquitin ligases have been described based on the presence of specific domains, one of which carries U-box or F-box domains (Hershko and Ciechanover 1998; Heyninck and Beyaert 2005). Thus the L. pneumophila U-box and F-box proteins might modulate the eukaryotic ubiquitination machinery. F-box proteins assembled into SCF ubiquitin ligase complexes determine which substrate will be targeted for ubiquitination and subsequent proteolysis by the proteasome. Thus it is tempting to assume that L. pneumophila is able to tag proteins for ubiquitination, which then in turn are degraded by the proteasome. Four proteins with homologies only to eukaryotic proteins were identified for the first time in a prokaryotic genome: a sphingosine-1-phosphate lyase, a sphingosine kinase and two secreted apyrases, which are conserved in all three L. pneumophila genomes (Table 1.2A). In eukaryotes, sphingosine-1-phosphate lyase and sphingosine kinase are part of the sphingomyelin degradation pathway.

10


Sphingosine kinase phosphorylates sphingosine, a catabolite of ceramide, into sphingosine-1-phosphate, which is cleaved irreversibly by sphingosine1-phosphate lyase. As the balance of both ceramide and sphingosine-1-phosphate is important for inducing authophagy or apoptosis (Reiss et al. 2004), the presence of this protein in the Legionella genomes suggests that Legionella may influence the autophagy machinery, which recognizes Legionella phagosomes for lysosome delivery (Amer and Swanson 2005; Swanson and Fernandez-Moreira 2002). Recently, proteins homolog to sphingosine-1-phosphate lyase have been identified in other prokaryotic genomes like Symbiobacterium thermophilum, Erythrobacter litoralis, Burkholderia thailandensis and Burkholderia pseudomallei. Enzymes not identified in any other prokaryote to date are two apyrases (Table 1.2A). These enzymes have been isolated in authophagy vacuoles (Biederbick et al. 1999) and may thus influence the L. pneumophila phagosome by decreasing the concentration of NTPs and NDPs. Serine/threonine protein kinases (STPKs) are known to be essential virulence factors in other bacteria. They were shown to be, e.g., translocated into the host cells where they counteract the host defense by interfering with eukaryotic signal transduction pathways (Barz et al. 2000; Hakansson et al. 1996) or to be secreted into macrophage phagosomes, inhibiting phagosome–lysosome fusion and promoting intracellular survival (Walburger et al. 2004). The L. pneumophila strains encode three eukaryote-like STPKs (Table 1.2B). Multiple sequence comparisons of kinase domains from L. pneumophila strain Paris and other prokaryotic and eukaryotic STPKs cluster two of these STPKs (Lpp2626/Lpl2481/Lpg2556 and Lpp1439/Lpl1545/Lpg1483) within the group of eukaryotic STPKs, closest to STPKs from Entamoeba histolytica (Cazalet et al. 2004). This suggests that the L. pneumophila STPK may also modulate eukaryotic signal transduction mechanisms as well as host-cell trafficking pathways. The importance and specificity of these proteins for L. pneumophila is underlined by the results obtained from genome comparisons. In order to verify whether the presence and wide variety of these eukaryotic-like proteins is Legionella specific the motif search was conducted on 11 additional bacterial genomes. For these comparisons we selected C. burnetti, the prokaryote, evolutionary the most closely related to L. pneumophila, other intracellular pathogens like Shield flexneri and Listeria monocytogenes, other bacterial pathogens involved in pulmonary infection like Chlamydia pneumoniae, Pseudomonas aeruginosa or Mycobacterium tuberculosis, bacteria also present in the aquatic environment and capable of colonizing amoebae like Salmonella typhimurium and Escherichia coli K12 as reference genome (Table 1.3). Compared to L. pneumophila, none of these genomes encodes the same variety and number of proteins-containing motifs found preferentially in eukaryotes. With the exception of the ankyrin repeat family which is the most abundant in Wolbachia pipientis (Wu et al. 2004) and serine-threonine protein kinases, which are more abundant in M. tuberculosis (Cole et al. 1998), Legionella is the organism with the highest number of proteins with eukaryotic-like domains (Table 1.3).


11

TABLE 1.3. Distribution of eukaryotic-like domains in the genome of different pathogens.

L. pneumophilaParis L. pneumophilaLens L. pneumophilaPhiladelphia 1 Coxiella burnetii Chlamydia pneumoniae Pseudomonas aeruginosa Escherichia coli Listeria monocytogenes Salmonella typhimurium Shield flexneri Mycobacterium tuberculosis Bartonella henselae Wolbachia pipientis Rickettsia prowazekii

F-box proteins 3 2 2

Ser/thr protein kinases 3 3 3

Sec7 domain 1 1 1

Ankyrin domain 20 17 13

SET domain 1 1 1

U-box proteins 1 0 1

0 0

1 2

0 0

16a 0

0 1

0 0

0

2

0

3

1

0

0 0

0 1

0 0

0 0

0 0

0 0

0

0

0

0

0

0

0 0

0 8b

0 0

1 0

0 0

0 0

0 0 0

0 0 0

0 0 1

0 23c 3

0 0 0

0 0 0

a

13 reported in Seshadri et al. (2003) 11 described in Cole et al. (1998) c 23 reported in Wu et al. (2004) b

Thus the genome sequence reflects the co-evolution of Legionella with its eukaryotic hosts, e.g., freshwater amoebae, and suggests that the different eukaryotic-like proteins are important for the intracellular life cycle of L. pneumophila, conferring probably in part its capacity to subvert host functions and to modulate them to its advantage.

2.2. Putative Virulence Factors as Deduced from Sequence Analysis In addition to the eukaryotic-like proteins, proteins with homology to virulence factors of other organisms were identified through sequence analysis. The three sequenced L. pneumophila strains encode a protein homologous to the enteropathogenic E. coli (EPEC) virulence regulator BipA. These three Legionella proteins (lpp2875, lpl2737, lpg2822) are identical and show 68% similarity to the E. coli BipA protein. The Legionella bipA gene has 43% GC content, which is significantly higher than that of the rest of the chromosome (38%), indicating perhaps horizontal gene transfer. However, the BipA protein is a member of the ribosomebinding GTPase superfamily that is widely distributed in bacteria and plants. In EPEC, BipA is thought to function high up in diverse regulatory cascades. It was

12


shown to regulate the espC pathogenicity island and a type III secretion system specified by the locus of enterocyte effacement (LEE) and flagella-mediated cell motility (Farris et al. 1998; Grant et al. 2003). In L. pneumophila neither a homolog of the EspC toxin nor a type III system are present; however, BipA may be involved in regulation of flagellum expression. In a global gene expression analysis of L. pneumophila in its natural host Acanthamoeba castellani, we showed that bipA gene expression is growth phase dependent as its expression is between two- and threefold decreased in stationary phase with respect to exponential growth (Brüggemann et al. 2006). This is in accordance with a recent report about gene expression in Pseudomonas putida (Yuste et al. 2006). As flagella expression is also growth phase dependent, the hypothesis that BipA is involved in this complex regulation is possible. A gene encoding a putative inner membrane efflux protein similar to the virulence factor MviN is present in the three strains with strain Philadelphia 1 encoding two orthologs (lpg1087, lpg2635). Although present in both pathogenic and non-pathogenic strains, absence of this protein in S. typhimurium results in reduced virulence in a mouse typhoid disease model (Carsiotis et al. 1989; Rudnick et al. 2001) suggesting that it may be involved in virulence of L. pneumophila. Another interesting finding is the presence of three homologs of the factor for inversion stimulation Fis, which exists in only one copy in other genomes like C. buenetii, E. coli or Salmonella species. This DNA-binding protein is involved in many different processes including modulation of transcription. Recently this protein has been implicated in the control of virulence gene expression in E. coli and S. typhimurium (Keane and Dorman 2003; Kelly et al. 2004; Osuna et al. 1995) and biofilm formation in enteroaggregative E. coli (Sheikh et al. 2001). As the concentration of the Fis protein varies tremendously between exponential and stationary phase due to the stringent response, these proteins could be implicated in differential expression of virulence genes during the Legionella cell cycle. Legionella pneumophila strain Paris encodes a specific protein, which bears hallmarks of type-V secretion systems or autotransporters. It is composed of three major domains: an N-terminal leader peptide for secretion across the inner membrane, a C-terminal domain that forms a pore in the outer membrane thus processing the passenger domain to the cell surface (Desvaux et al. 2004). Like the E. coli autotransporters AIDA-I and Ag43, the L. pneumophila autotransporter contains hemagglutinin repeats, which usually confer the protein a function in cell-to-cell aggregation (Benz and Schmidt 1992; Klemm et al. 2004). However the known RGD interaction motif is absent in the L. pneumophila autotransporter. Thus, another interaction motif might exist and confer a role in adherence to mammalian cells and/or autoaggregation during biofilm formation. This autotransporter was probably acquired through horizontal gene transfer as it is suggested by its GC content of 41% higher than the average of 38% and the presence of many remnants of insertion sequences upstream.


13

2.3. Secretion Machineries—Presence of Particular Many and a Wide Variety Legionella pneumophila has a rather exceptional number and wide variety of secretion systems for efficient and rapid deliverance of effector molecules into the phagocytotic host cell underlining the importance of protein secretion for this pathogen. The two major ones, known to be involved in virulence are the Dot/Icm type-IV secretion system and the lsp type II secretion system. Legionella pneumophila and C. burnetii are the only bacterial pathogens known to encode a type-IVB secretion system similar to the Tra/Trb system of IncI plasmids. This type-IVB secretion system is called icm (intracellular multiplication) (Marra et al. 1992) and/or dot (defective organelle trafficking) (Berger and Isberg 1993) system. It is required for intracellular growth in human macrophages as well as in amoebae and for intracellular trafficking. This macromolecular complex is encoded by 25 genes located on two genomic regions that are conserved and located in the same chromosomal location in strains Paris, Lens and Philadelphia 1. Morozova and colleagues (Morozova et al. 2004) showed by sequence comparison and DNA/DNA hybridization that the organization of this region is conserved among 18 L. pneumophila and 17 other Legionella species tested. Hybridization studies using a Legionella macro-array carrying these 25 genes further proved their presence and conservation among 180 tested L. pneumophila strains of all serotypes and different origins (Cazalet et al. 2005a) underlining again that the Dot/Icm type IV system is essential for the life as well as pathogenicity of Legionella. Different substrates of the Dot/Icm secretion system have been identified like RalF, which is required for the localization of ARF on phagosomes containing L. pneumophila (Nagai et al. 2002), LidA implicated in establishing a replicative niche (Conover et al. 2003; Derre and Isberg 2005), LepA and LepB involved in the release of Legionella from the vacuole after intracellular multiplication (Chen et al. 2004). Recently a large number of candidate effector proteins, designated SidA-G (substrate of Icm/Dot transporter) using a two-hybrid screen, and subsequent screening using a Cre/loxP-based system were identified (Luo and Isberg 2004). Interestingly, most of the Sid proteins have one or more paralogs present in the L. pneumophila genomes, and each of the three sequenced genomes have a slightly different array of Sid proteins. Furthermore, many effector proteins show no sequence similarity to proteins from other organisms, which indicates the uniqueness of the mechanism by which L. pneumophila subverts eukaryotic hostcell functions. The second type IV secretion system encoded by L. pneumophila is a type-IVA system similar to the Agrobacterium tumefaciens Vir system. It is dispensable for intracellular growth in both macrophages and amoebae (Segal et al. 1999), but it is implicated in host-cell infection by L. pneumophila at 30°C (Ridenour et al. 2003) and it can also mediate conjugational DNA transfer (Segal et al. 1999). To date L. pneumophila is the only intracellular pathogen known to encode a type-II secretion system. It is highly conserved in the three sequenced genomes

14


(protein identities of 96–100%) and seems also to be conserved throughout the genus Legionella (Rossier and Cianciotto 2001). The lsp secretion system promotes the ability of L. pneumophila to infect both protozoan and macrophage hosts, to grow in the mammalian lung (Rossier and Cianciotto 2001) and to grow at low temperatures (Soderberg et al. 2004). Legionella pneumophila possesses several additional secretion systems like a type I secretion system called lss, which seems to be specific to L. pneumophila (Jacobi and Heuner 2003), a sec apparatus, which is complete with the exception of secG and a twin arginine translocation (TAT) pathway. In contrast to the usual organization of tatA, tatB and tatC in an operon, only the tatA and tatB genes are co-transcribed and tatC is in a different location of the L. pneumophila chromosome. This organization is conserved in all three genomes. In strains Paris, Lens and Philadelphia 1, the tatAB operon and the tatC gene are located 24 kb, 41 kb and 27 kb apart, respectively. However, the flanking regions are, despite the different distance between the tatAB and tatC genes, homologous in the three strains. The TAT secretion system is implicated in growth under low iron conditions, growth within macrophages in an Lsp-independent manner (Rossier and Cianciotto 2005). In addition to these conserved secretory pathways, strain Paris encodes a type-V secretion system specific to this strain.

3. Metabolism as Deduced from the Genome Sequence Legionella pneumophila is a quite fastidious organism to grow. The bacterium preferably utilizes proteinaceous substrates. This is reflected in the genome sequence, as a large set of oligopeptide and amino acid uptake and degradation systems is present. Legionella pneumophila requires arginine, isoleucine, leucine, valine, methionine, serine and threonine for growth. Genome analysis identified the biosynthetic pathways for all remaining amino acids but also for arginine. Thus, the auxotrophy for six amino acids but not for arginine is in agreement with the genome analysis. Together with many amino acid permeases, a broad range of secreted proteases for serine, cysteine and metal type ones are encoded by the genomes. In particular, besides the P. aeruginosa elastase homolog ProA three secreted paralogous metalloproteases are present. The presence of more than 40 peptidases in each genome suggests the breakdown of proteins to amino acids by Legionella. This suggests that besides free amino acids, proteins and peptides may also be an important source of nutriment for L. pneumophila. In contrast, although harboring the complete Embden–Meyerhof as well as the Entner–Doudoroff pathway, systems for sugar uptake seem to be rare; no PTSlike uptake systems are encoded by the genomes. However, some of the about 50 identified ABC-type transport systems might be involved in sugar uptake, since the bacterium possesses a few systems for the degradation of complex sugars, such as trehalase, polysaccharide deacetylase, glucoamylase, ␤-hexosaminidase and chitinases. The extracellular enzymes chitinase and a glucoamylase probably


15

used for the degradation of sugar polymers are similar only to eukaryotic enzymes. The glucoamylase gene clusters encode enzymes of the glycolysis and a sugar permease, suggesting a significant role in catabolism. The pentose phosphate pathway and glycolysis are complete although the fructose 1–6 diphosphate aldolase is similar to eukaryotic enzymes. The key regulatory enzyme of glycolysis, phosphofructokinase (pfk) of L. pneumophila, seems to be bifunctional as it is not of the ATP-dependent but the PPi-dependent type. The widely distributed ATP-PFK catalyzes an irreversible catabolic reaction, the phosphorylation of fructose-6-phosphate to fructose-1,6-bisphosphate, while the PPi-PFK, also called PPi-phosphofructokinase (PFP), catalyzes the same reaction in a reversible way and can thus function both in glycolysis and gluconeogenesis (Baptestea et al. 2003). The Legionella genomes also encode several enzymes for the utilization pathway for myo inositol, which may interfere with the host-cell signaling mediated by this intracellular messenger. The complete metabolic pathways for biotine, riboflavin, folate, nicotine amine and heme are present. However, the ubiquinone biosynthesis pathway in L. pneumophila is different to the one described for E. coli and may correspond to an uncharacterized biosynthetic pathway also present in P. aeruginosa. Legionella pneumophila employs an extensive aerobic respiratory chain consisting of NADH dehydrogenase, cytochrome-dependent succinate dehydrogenase, ubiquinol-cytochrome c reductase and four terminal oxidases, which guarantee the capacity to cope with changing oxygen tensions (an aa3 cytochrome, two bd-type quinol cytochrome and an o-type quinol cytochrome oxidase). The o-type oxidase, encoded by the cyo operon is present in strain Paris but is missing in strains Lens and Philadelphia 1. Systems involved in anaerobic respiration are apparently absent. Two ATP synthases are encoded by the genomes, an F0F1-type ATP synthase of ␥-proteobacteria present in all three genomes and a second one with highest similarity to systems of archaea and marine bacteria present only in strains Paris and Philadelphia 1. At least four sodium/proton antiporters seem to be involved in the interchangeability of the proton and the sodium gradient across the cytoplasmic membrane, raising the question if a sodium motive force is used for cellular activities. In this respect, the presence of a key component of sodium-type polar flagellar motors, MotY, as well as two significantly different MotAB gene clusters lead to the assumption that motility is powered by sodium as well as proton motive forces.

4. Clues to Adaptation—Regulation and Signal Transduction Pseudomonas aeruginosa and L. monocytogenes, which are both able to adapt to the host environment and to multiply and survive in a wide variety of environments outside a host contain large sets of regulatory proteins with 8.4% and 7.3% of the genome, respectively, dedicated to regulatory proteins (Stover, 2000 pp. 150; Glaser, 2001 pp. 149). In contrast, L. pneumophila is an intracellular

16


pathogen that does not seem to be able to replicate in the environment outside the host. Consistent with this lifestyle the regulatory repertoire is rather small. Genome analysis identified 92, 79 and 106 transcriptional regulators in strains Paris, Lens and Philadelphia 1, respectively. This represents only a little more than 3% of the predicted genes. Each L. pneumophila genome encodes six putative sigma factors, the homologs of rpoD (the major sigma factor), rpoH, rpoS, rpoN, fliA and the ECF-type sigma factor rpoE. Furthermore one sigma 54 (RpoN) modulator, sigma factor-dependent enhancers (FleQ, FleR, PilR) and the anti-sigma factors FlgM and anti-sigma factor B are encoded by all three L. pneumophila strains. The number of two-component systems (13 histidine kinases and 13 response regulators) is also substantially lower compared to that of free-living bacteria like E. coli (35) or P. aeruginosa (100) but conserved in all three genomes. The most prominent classes of transcriptional regulators in L. pneumophila are LysR-type and phage-like repressors. The abundance of the latter class probably indicates that the genome is subject to continuously ongoing phage-mediated horizontal transfer. However, interestingly, in none of the three genomes sequenced, a complete prophage was identified. Regulators of the GGDEF/EAL family are abundant in L. pneumophila. The three strains encode 24, 21 and 21 regulators of this type, respectively (Table 1.4), TABLE 1.4. GGDEF/EAL regulators identified in the three L. pneumophila genomes. Paris lpp0029 lpp0087 lpp0127 lpp0219 lpp0220 lpp0299 lpp0351 lpp0352 lpp0440 lpp0809 lpp0891 lpp0942 lpp0952 lpp1114 lpp1170 lpp1311 lpp1425 lpp1475 lpp2071 lpp2324 lpp2355 lpp2477 lpp2695 lpp2708

Gene name Lens lpl0030 lpl0075 – lpl0219 lpl0220 lpl0283 – lpl0329 lpl0416 lpl0780 lpl0860 lpl0912 lpl0922 lpl1118 lpl1176 lpl1308 lpl1559 lpl1508 lpl2061 lpl1054 – – lpl2567b lpl2581

Philadelphia lpg0029 lpg0073 – lpg0155 lpg0156 lpg0230 – lpg0277 lpg0373 lpg0744 lpg0829 lpp0879 lpp0891 lpg1114 lpg1168 lpg1357 lpg1469 lpg1518 lpg2132 lpg1057 lpg1025 – lpg2642 lpg2655

Domains 3 PAS, GGDEF, EAL GGDEF, EAL EAL, COG1639 PAS, GGDEF EAL CHASE3, GAF, GGDEF EAL Response-Regulator, PAS, GGDEF GGDEF GGDEF GGDEF, EAL, HAMP GGDEF 2 PAS, GGDEF, EAL PAS, GGDEF, EAL GGDEF, EAL PAS, GGDEF, EAL EAL PAS, GGDEF, EAL GGDEF, EAL GGDEF, EAL GGDEF GGDEF 2 PAS, GAF, GGDEF, EAL GGDEF

TMa helices – 2 – – 1 1 – – 5 – 3 5 – – 9 7 2 13 9 4 5 1 – 6


17

similar to many other bacteria, like Vibrio cholerae (41), P. aeruginosa (33), Wolinella succinogenes (26), and E. coli (19) (Aldridge et al. 2003; Baar et al. 2003). These regulators contain different N-terminal signaling domains and a C-terminal output domain, which is composed of two subdomains, GGDEF (DUF1) and EAL (DUF2) (Tal et al. 1998). Those domains modulate the intracellular level of cyclic-di-GMP, a recently discovered secondary messenger in the bacterial cell, which governs multicellular behavior such as the switch from sessility (e.g., in a biofilm) to motility (e.g., swarming) in the pathogenic organisms Salmonella enterica serovar Typhimurium, the nosocomial pathogen P. aeruginosa and the commensal species E. coli (Simm et al. 2004). Moreover, an EAL domain protein in S. enterica is involved in the resistance to oxidative stress and in the ability to kill macrophages (Hisert et al. 2005). Thus, EAL/GGDEF proteins could have a global role in governing the developmental differentiation of L. pneumophila.

5. Comparative Genomics of Legionella pneumophila 5.1. The Specific Gene Complements of L. pneumophila Paris, Lens and Philadelphia Legionella research is in a privileged situation, having complete genome sequences for three different isolates of the species L. pneumophila available. Whole genome comparisons allow now to define specific and common features. According to these comparisons, the L. pneumophila genomes, similar to many other bacterial species, consist of a conserved core part and a so-called “flexible gene pool.” The conserved core region is required for essential functions such as protein biosynthesis, the cell envelope and major metabolic pathways. In the flexible gene pool, plasmids as well as genomic and pathogenicity islands represent the major genetic elements. Furthermore, IS-elements and transposases were identified in the flexible gene pool as well as small DNA-pieces, termed “islets.” Comparison of strains Paris, Lens and Philadelphia 1 identified about 2500 genes as the conserved backbone and about 300 genes (⬃10%) in each genome as strain specific, constituting thus the flexible gene pool (Figure 1.3). Given the fact that the three analyzed strains belong to the same species and same serogroup, this diversity is quite high. Most of the strain-specific genes code proteins of unknown function; however, some proteins may be related to strain-specific niche adaptation or may code for specific virulence functions. The specific genetic equipment of strain Paris contains a number of regulators (two CsrA homologs, 11 transcriptional regulators), five additional ankyrin-repeat, one specific U-box domain protein and two eukaryotic-like proteins (Table 1.2A and Table 1.2B) as well as several restriction modification genes (DNA modification methylases, endonucleases), a specific

18

Christel Cazalet and Carmen Buchrieser L. pneumophila Paris 3077* * : plasmids are not considered

313 (10%)

134

130 2500

L. pneumophila Philadelphia 2953

283 (10%)

257 (9%) 40

L. pneumophila Lens 2931 *

FIGURE 1.3. Diagram showing the core genome and the unique gene complement of strains L. pneumophila Paris, Lens and Philadelphia. Orthologous genes were defined by reciprocal best-match FASTA comparisons. The threshold was set to a minimum of 80% sequence identities and a ratio of the length of 0.75–1.33.

o-type cytochrome ubiquinol oxidase and a type V secretion system (autotransporter). Strain Lens contains four specific transcriptional regulators and three specific proteins with eukaryotic domains (Table 1.2A), two of which are ankyrin repeat proteins. Except four specific transcriptional regulators, Strain Philadelphia 1 codes two specific ankyrin repeat and one F-box domain protein. Strain Philadelphia 1 codes, in addition to the three peptide methionine sulfoxide reductase regulators msrA that are conserved in all strains, three additional orthologs msrA1,2,3 on its specific 65-kb pathogenicity island. msrA2 and msrA3 are homologous to msrA and msrB encoding antioxidant repair enzymes, characterized as virulence determinants in E. coli, Neisseria gonorrhea and Mycoplasma genitalium (Dhandayuthapani et al. 2001; Moskovitz et al. 1995; Skaar et al. 2002). In the same island the Philadelphia 1 specific magA gene, coding a protein putatively implicated in the maturation of the cyst-like mature intracellular form, is present (Hiltz et al. 2004). Furthermore, as mentioned before, each of the three sequenced genomes have a slightly different array of Sid proteins, candidate effectors of the Dot/Icm type IV secretion system (Luo and Isberg 2004).

5.2. Genomic and Pathogenicity Islands Like many bacterial species, the L. pneumophila genomes carry several pathogenicity island like regions. One example is the region encoding the lvh type-IV secretion system which is dispensable for intracellular growth in both macrophages and amoebae (Segal et al. 1999), but implicated in host-cell infection by L. pneumophila at 30°C (Ridenour et al. 2003) and which can also


19

mediate conjugational DNA transfer (Segal et al. 1999). The lvh cluster itself is highly conserved in all three sequenced genomes. However, the flanking DNA regions are specific to each strain in size as well as in the genetic content. In strain Paris the lvh region is flanked by 11 kb and 22 kb, in strain Lens by 5.8 kb and 30 kb and in strain Philadelphia 1 by 14 kb and 30 kb. This region is inserted in the same tRNA in strains Lens and Paris but in an Arg tRNA in another chromosomal location in strain Philadelphia 1. The lvh region has in addition the particular capacity to exist in an integrated and an excised form as multi-copy plasmid of 36 kb in size in strain Paris and of 45 kb in strain Philadelphia 1. This was not observed in strain Lens; however, a 28bp perfect repeat is present in the flanking regions, suggesting that it is also able to excise. This element is probably of plasmid origin as it has a higher GC content (43%) than the rest of the chromosome (38%) and it carries some plasmid related genes. A similar element, contributing to phase variation that can also exist in a plasmid as well as integrated form, was previously identified in strain OLDA (Luneberg et al. 2001). The identification of the exact mechanism leading to the excision and integration of these elements will help to understand the adaptation and versatility mechanisms of Legionella. A second chromosomal region fulfilling the typical characteristics of pathogenicity islands is a 40 kb region encoding several efflux pumps induced in contact with the host cell. It is apparently dedicated to detoxification and proper metal ion balance within the bacteria (Rankin et al. 2002). In the three sequenced strains, the cluster coding the efflux pumps is preceded by genes homologous to lvrABC and is located between conserved regions encoding mostly proteins of unknown functions. Typically for pathogenicity islands these regions are interrupted by several insertions specific to each strain and are often flanked by integrase genes or insertion sequences. In strains Philadelphia 1 and Lens this large island of 135 kb and 87 kb, respectively, is located on the same chromosomal location and is flanked by a Thr tRNA gene. In strain Paris it is forming a large island of 130 kb present in a different chromosomal location and is flanked by a Met tRNA gene. The 65 kb pathogenicity island (LpPI-1), described previously as specific for strain Philadelphia 1 (Brassinga et al. 2003), is indeed absent from strains Paris and Lens. However, genome comparison by hybridization of 180 Legionella strains showed that this island is also present in other L. pneumophila strains as well as in strains of other Legionella species (Cazalet et al. 2005a). In strain Philadelphia 1, this island is inserted in a Val tRNA gene, it contains putative virulence factors already mentioned before, mobile elements and an additional cluster of genes with higher G ⫹ C content than the rest of the chromosome, encoding homologs of Tra proteins. The Tra region shows in average 55% similarity to the Tra proteins associated with the F plasmid of E. coli. The LpPI-1 tra gene homologs are arranged identically to those of the F plasmid, with the exception of traM, traY, and traX, which are missing in L. pneumophila. Comparative analysis of the borders of LpPI-1 in eight additional L. pneumophila strains lacking the core region of this island revealed pronounced genetic variability among

20

Christel Cazalet and Carmen Buchrieser Lens

Lys tRNA

Paris

Philadelphia Weakly similar to eukaryotic proteins Similar to bacteriophage proteins Conserved regions between the three genomes Integrase Regulator

FIGURE 1.4. Genetic organization of a putative pathogenicity island specific of L. pneumophila strain Paris and its chromosomal location as compared to strains L. pneumophila Lens and Philadelphia. The figure was created by using ACT (Artemis Comparison Tool) software (http://www.sanger.ac.uk/Software/ACT/).

the strains, suggesting that this region represents a “plasticity” or hypervariable zone (Brassinga et al. 2003). This hypothesis is reinforced by the comparison of this genomic region in the three sequenced strains. In strains Paris and Lens small insertions of 25 kb and 19.3 kb, respectively, which code mainly unknown proteins but also two ankyrin repeat proteins, a homolog of SidE, a regulatory protein and four transposases, are present. An islet of 11.3 kb inserted in a Lys tRNA gene is present only in strain Paris (Figure 4). Interestingly, this island contains an integrase, two phage-related genes and 14 genes coding proteins with some similarity to eukaryotic proteins. The G ⫹ C content of this region has a mosaic composition varying between 28 and 41% with the eukaryotic-like proteins showing mainly low G ⫹ C content of 30% in average. This region might be in favor of an acquisition of some eukaryotic-like proteins through horizontal gene transfers mediated by bacteriophages. Others, however, might have evolved by convergent evolution. Some of these pathogenicity/genomic islands of L. pneumophila are present in the three genomes at different locations or only in part. Figure 1.5 illustrates the genome rearrangements and plasticity in indicating these flexible regions with respect to their size and chromosomal location as compared to the three sequenced genomes.

5.3. Plasmids of Legionella Two of the three sequenced L. pneumophila strains carry plasmids that might play a role either in adaptation to the environment or in virulence as both encode putative virulence factors, mobile genetic elements and antibiotic resistance genes. In strain Paris a 132 kb plasmid and in strain Lens a 60 kb plasmid was identified, both of which seem to be single copy plasmids. These plasmids contain a 24 kb


21

FIGURE 1.5. Regions of plasticity identified in the L. pneumophila genomes and their chromosomal locations in the three sequenced strains. Outer circle: Strain Paris; main specific regions are indicated in light grey; Central circle: Strain Lens, main specific regions are indicated in dark grey; Inner circle: Strain Philadelphia 1, main specific regions are indicated in grey. dot/icm, Dot/Icm type IV secretion system; lvh, Lvh type IV secretion system; efflux cluster; efflux pumps located on a plasticity zone of 130 kb (strain Lens), 135 kb (strain Philadelphia 1) and 87 kb (strain Lens).

and 30 kb gene cluster, respectively, encoding homologs of Tra proteins (Figure. 1.6). Furthermore several mobile elements like transposases and phage-related proteins are present and about half of the genes code proteins of unknown function. Both plasmids also encode a paralog of CsrA—a protein known to act as repressor of transmission traits of L. pneumophila and as activator of replication (Molofsky and Swanson 2003), revealing the question whether these plasmids are implicated in virulence. The plasmid identified in strain Paris carries many genes coding proteins probably conferring antibiotic resistances, like a betalactamase, two spectinomycin 3⬘ adenylyltransferases or an aminoglycoside 3⬘ phosphotransferase. Aminoglycoside phosphotransferase (3⬘)-IIIa (APH) is a bacterial kinase that confers antibiotic resistance to many pathogenic bacteria and shares structural homology with eukaryotic protein kinases. The identified betalactamase is 93% similar to a previously identified D beta-lactamase determinant isolated from Legionella (Fluoribacter) gormanii ATCC 33297(T) where it was shown to transfer resistance to oxacillin, methicillin, penicillin G, ampicillin, carbenicillin, piperacillin and also for cefazolin and nitrocefin, oxyimino cephalosporins and aztreonam (Franceschini et al. 2001). Furthermore nine acetyltransferase genes,

22

Christel Cazalet and Carmen Buchrieser 0

A

B

120

0

15

egion Tra-r

105

Plasmid Legionella pneumophila strain Paris 131.9 kb

30 45

Plasmid Legionella pneumophila strain Paris 59.8 kb

15

90 45

ion

reg

Tra 30 75

60

FIGURE 1.6. Circular map of the plasmid present in strain Paris (A) and strain Lens (B). For both plasmids from the outside—circle 1: Plasmid genes on the ⫹ and – strands, respectively. Black, Tra-region (traTLEKBVC, trbI, traWU, trbC, traNF, trbB, traHGDI); circle 2: G/C bias (G ⫹ C/G-C); 3: G ⫹ C content of the plasmid with ⬍32.5% G ⫹ C short length, between 32.5% and 44.1% medium length and with ⬎44.1% G ⫹ C in long lines.

two of which belonging to the GCN5-related N-acetyltransferase (GNAT) superfamily, are present which could either be implicated in detoxification, drug resistance or gene regulation. Interestingly, 6 kb of the Paris plasmid contain seven genes coding for proteins with 91–98% protein similarity to proteins coded by a plasmid previously identified in a L. longbeachae isolate. Most of the proteins code for unknown functions, but two code for a two-component system, lrpR/lskS, that was shown to be implicated in virulence of this species (Doyle and Heuzenroeder 2002). In contrast, the plasmid identified in strain Lens contains mainly genes coding for proteins of unknown functions. The role of specific plasmids in virulence of Legionella has not clearly been demonstrated, but a correlation between virulence in a mouse model and the presence of plasmids (Bezanson et al. 1994) and a higher persistence in the environment of strains containing plasmids (Brown et al. 1982) has been reported. Transfer of plasmids from a L. pneumophila donor to another L. pneumophila isolate or another Legionella species may be mediated by the Dot/Icm type-IV secretion system (Vogel et al. 1998). The identification, characterization and analysis of the functional role of Legionella plasmids might add to the understanding of versatility, adaptation and virulence mechanisms of this pathogen.

5.4. Variability at the Gene Level The plasticity of the Legionella genomes is not only due to insertions, deletions or rearrangements of genomic islands of different size or due to plasmids, but plasticity was also identified for single genes that seem particularly variable.


23

An example for plasticity at the gene level is the rtxA gene. This gene was first described in strain L. pneumophila AA100 where it is located next to a gene named arpB. In strain Paris, the rtxA and arpB genes are fused and are followed by a region of about 17 kb of repetitive DNA organized in approximately 30 highly conserved tandem repeats. The same organization is observed in strain Lens; however, two kinds of repeats, which are completely different from those observed in strain Paris, are present. In strain Philadelphia 1, arpB is missing and a frameshift disrupts the fusion with the repeat region. The tandem repeats are also highly conserved but are again a new repeat type. Probably variations in the number and sequence of these repeats contributes to versatility and perhaps also to virulence of Legionella. Another example is the dotA gene that is part of the Dot/Icm type IV secretion system. The comparison of this system, in different Legionella species and L. pneumophila serogroups, reveals a complicated evolutionary history and identifies an extensive polymorphism and mosaic structure of the dotA gene due to several intragenic recombination events. DotA is a secreted protein implicated in survival within macrophages and inhibition of phagolysosome fusion by Legionella. Thus it is supposed that variability and rapid evolution of DotA creates antigenic variations for adaptation to certain environmental niches (Ko et al. 2003a, 2003b). Variation on the gene level is also seen in otherwise conserved regions among different Legionella species, as reported by Feldman and colleagues (Feldman and Segal 2004). The analysis of sequence variability of two essential components of the Dot/Icm system: icmQ, coding the pore-forming protein IcmQ and icmR, coding its regulator IcmR in 29 Legionella species revealed that the organization of this region is highly conserved, but that it contains a favorable site for gene variation. In the place where the icmR gene was expected to be located, other open reading frames that are non-homologous to each other or to any entry in the GenBank database were found (migAB in L. micdadei and ligB in L. longbeachae). This led to the identification of a hypervariable gene family named fir for functional homologs of icmR whereas icmQ is conserved. The only exception is a small variable region important for the interaction between the two proteins. Supposedly, the Fir proteins and the variable part of IcmQ coevolved enabling adaptation to the environment and survival in various protozoan hosts.

6. Comparative Genomics—Legionella, Coxiella and Other Intracellular Pathogens 6.1. Genome Comparison of Legionella pneumophila and Coxiella Burnetii Comparative genome analysis can be used to identify species-specific genes and gene clusters, and analysis of these genes can give insight into the mechanisms involved in specific host–pathogen interactions. We thus compared the genomes of

24


L. pneumophila with that of the evolutionary most closely related species, C. burnetii. Coxiella burnetti is an obligate intracellular pathogen that, like Legionella, thrives on both macrophages and amoebae. Thus the comparison of these genomes should allow the identification of common pathways and mechanisms probably important for intracellular trafficking and the specificity of these organisms. Indeed, both organisms are vacuolar pathogens; they have a biphasic life cycle and alternate between a transmissive and a replicative phase and both of them are able to delay phagosome–lysosome fusion early in infection. However, different to C. burnetti, L. pneumophila replicates in a vacuole surrounded by endoplasmic reticulum. A global comparison identifies approximately 42% of the Legionella genes as shared with those predicted in Coxiella. As expected from their genetic and cell biological similarities some virulence factors are conserved in both organisms. Indeed, Coxiella encodes two homologs of the global regulator csrA that is important in Legionella for regulation of its biphasic life cycle, and the proteins enhABC, which are involved in entry of L. pneumophila into host cells. Interestingly, Coxiella and Legionella are the only two bacteria known to date to use a type-IVB secretion system (Dot/Icm in Legionella) for pathogenesis. The organization of the gene cluster is almost identical in both organisms and with the exception of the icmR gene the Dot/Icm secretion system of Legionella is conserved in Coxiella (Segal et al. 2005). However, interspecies complementation of L. pneumophila dot/icm mutants by homologous proteins of Coxiella showed that there exists only partial functional similarity between the two systems. Twentytwo effectors and potential effector proteins were identified in L. pneumophila. Surprisingly, none of them has a homolog in C. burnetii suggesting different mechanisms of subverting host functions for these two pathogens. In contrast to L. pneumophila, which shows high genome redundancy, C. burnetti seems to have undergone genome reduction as many intracellular pathogenic microorganisms, which are strictly host-adapted (Seshadri et al. 2003). However, some large protein families are conserved in both organisms. An example is the ankyrin protein family. Legionella pneumophila encodes 13–20 ankyrin repeat proteins and C. burnetii encodes 13, despite its smaller genome size of 1.99 Mb. Similarly, the recently identified Pht (phagosomal transporters) subfamily of MFS transporters (Sauer et al. 2005) has nine to eleven members depending on the sequenced strain of L. pneumophila, and C. burnetii encodes also nine transporters of this class. In Legionella, one of these MFS transporters, named PhtA, was shown to mediate the differentiation from transmissive to the replicative form. It was suggested that this transporter subfamily allows the vacuolar pathogens Legionella and Coxiella to assess phagosomal nutrient supply before entering the replicative phase (Sauer et al. 2005).

6.2. Genome Redundancy—Gene Family Expansion in Legionella Genome sequencing and comparison revealed expansion of some gene families as well as high functional redundancy in L. pneumophila. One of the largest classes


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of proteins are transporters like in many genomes; however, in Legionella multidrug efflux pumps for heavy metals and other toxic substances are highly represented. They could play an important role in adaptation to the environment and might also play a role in biocide resistance. Legionella also possess a prominent family of regulators with GGDEF-EAL domains (24 in strain Paris, 21 in strain Lens and 21 in strain Philadelphia 1), which are essential for virulence properties of many bacteria as they are involved in aggregation, biofilm formation or twitching motility. This protein family is also largely represented in many other bacteria; however, C. burnetii, the most closely related prokaryote to Legionella, encodes only one. Moreover, some virulence protein families contain several orthologs. For example, four, six and three (Paris, Lens, Philadelphia 1, respectively) paralogs of the major outer membrane protein Momp are encoded by Legionella. The metalloprotease ProA implicated in virulence has eight paralogs, and the enhanced entry protein EnhC has four paralogs. Furthermore three paralogs of the factor for inversion stimulation Fis as discussed in Section 2.2 are present in the three Legionella genomes. Similarly, many substrates of the Dot/Icm system such as the Sid proteins are part of large paralogous families with functional redundancy. This high functional redundancy of the L. pneumophila genomes provokes a question whose answer has both evolutionary and virulence implications: are all these paralogous proteins functionally redundant, or have they acquired sufficient specialization to enable adaptation to different niches like different protozoa that the bacteria may encounter? It seems that there is both specialization and redundancy in the functions of these proteins.

7. Comparative Genomics of Legionella pneumophila and the Genus Legionella—a Perspective Comparative genomics and related technologies are helping to unravel the molecular basis of the pathogenesis, host range, evolution and phenotypic differences of Legionellae. In Legionella research the area of genomics started with the determination of three complete genome sequences of clinical isolates of L. pneumophila. The availability of these three sequences opened the way to comparative and functional genomics and allows now to use new approaches like bioinformatics, micro-arrays and proteomics to gain functional information. Furthermore, comparison of pathogenic and apathogenic members of the genus Legionella should bring new insight into virulence functions important for disease in humans and into evolution of Legionellae. Thus further sequencing efforts are needed for in-depth comparative genomics. A second Legionella species is important for human disease, L. longbeachae, which accounts for 30.4% of community-acquired legionellosis cases in Australia and New Zealand (Yu, 2002 pp. 21). Other species like L. anisa, L. micdadei, L. dumofii or L. feeleii seem to be less virulent even though they relatively frequently colonize water distribution systems (Muder and Yu 2002), or have

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never been implicated in human disease like L. hackaliae. Recently, a first comparison of the completely sequenced L. pneumophila genomes with a partially sequenced L. anisa and L. longbeacheae isolate revealed a very high diversity, as only around 50% of the genome seems to be conserved among the three Legionella species (Cazalet et al. 2005b). Except these first preliminary comparisons, very little is known to date on the genomic level about the degree of the genetic diversity among different isolates of L. pneumophila of different serogroups or among different Legionella species. In particular, the relation between a specific gene content and virulence is unknown. However, this knowledge is a prerequisite for understanding differences in pathogenicity and the ecology of this organism and it is also the basis for the development of rational and accurate diagnostic tools. Importantly, additional sequence information will soon be available as a fourth L. pneumophila isolate, strain Corby, is being sequenced by the Institut for Molecular Biotechnology, IMB Jena, Germany. Furthermore, the determination of the genome sequences of L. hackeliae and L. mikdadei by the same Institution and that of an isolate of L. longbeachae by the Institut Pasteur, Paris, France, are nearing completion. The availability of these different genome sequences of Legionella will pave the way for in-depth comparative genomics and the identification of so far unknown virulence determinants.

Acknowledgments. We would like to thank many of our colleagues who have contributed in different ways to this research. In particular, P. Glaser for the many fruitful discussions, F. Kunst for his support and C. Rusniok for his help and never-ending patience with informatics problems or questions. This work received financial support from the Institut Pasteur, the Centre National de la Recherche and from the Consortium National de Recherche en Génomique du Réseau National des Genopoles, France. C. Cazalet is holder of a fellowship jointly financed by the Centre National de la Recherche, France, and VeoliaWater—Anjou Recherche.

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2 Iron Assimilation and Type II Protein Secretion Nicholas P. Cianciotto

1. Iron Assimilation 1.1. The Importance of Iron for Legionella pneumophila Iron is a key requirement for L. pneumophila replication (Reeves et al, 1981). As originally defined, the iron requirement for L. pneumophila was estimated to be 3–13 ␮M for minimal growth and ⬎20 ␮M for optimal growth (Reeves et al, 1983; Johnson et al, 1991; Mengaud and Horwitz, 1993). However, recent studies using chemically defined media indicate that the requirement is ⬍1 ␮M (James et al, 1995; Liles et al, 2000). In standard BCYE agar, iron is added in the form of ferric pyrophosphate, although ferric chloride, ferric nitrate, and ferrous sulfate can also be used. Upon incubation with 55FeCl3, L. pneumophila uptakes significant amounts of radiolabeled iron in an energy-dependent, protease-resistant process (Johnson et al, 1991). L. pneumophila also binds and uses hemin as a source of iron (O’Connell et al, 1996). As in other bacteria, L. pneumophila requires iron as a cofactor in enzymes, such as a superoxide dismutase and aconitase (Mengaud and Horwitz, 1993). Iron is especially relevant for Legionella pathogenesis. Much evidence signals that the ability of L. pneumophila to replicate in the mammalian host is dependent upon iron. First, human monocytes and macrophages treated with iron chelators do not support Legionella replication, and interferon-␥ inhibits bacterial growth by reducing available iron (Byrd and Horwitz, 2000; Viswanathan et al, 2000). Second, A/J mouse macrophages become permissive for Legionella infection following the addition of iron (Gebran et al, 1994). Third, a reduced ability of legionellae to establish intracellular infection is correlated with decreased levels of the receptor for transferrin, the key iron transport protein of the host cell (Byrd and Horwitz, 2000). Fourth, the introduction of iron into animals increases their susceptibility to infection. Fifth, legionellae grown under iron-deplete conditions exhibit reduced pathogenicity (James et al, 1995). The first genetic data on the importance of iron in Legionella was the identification of the transcriptional repressor Fur (Hickey and Cianciotto, 1994). L. pneumophila Fur is comparable in size and cross-reactive with Escherichia

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Nicholas P. Cianciotto

coli Fur. Its repressive activity is, as expected, highest in legionellae grown in iron-rich media (Hickey and Cianciotto, 1994, 1997). L. pneumophila Fur has an amino acid identity of over 54% and a similarity of over 72% to Fur from E. coli, Pseudomonas aeruginosa, and others (Hickey and Cianciotto, 1994). The promoter region of L. pneumophila fur contains sequences homologous to the Fur-binding site, suggesting that fur is auto-regulated in Legionella. The fur gene is conserved among Legionella species (Hickey and Cianciotto, 1994). Given an inability to insertionally inactivate L. pneumophila fur, Fur may be essential for Legionella aerobic growth. The importance of fur is substantiated by the identification of multiple iron- and Fur-regulated genes in L. pneumophila (Hickey and Cianciotto, 1997).

1.2. Legionella Siderophores 1.2.1. Early Negative Findings It was reported in 1983 that L. pneumophila does not make siderophores (Reeves et al, 1983). This conclusion was based largely on results from Arnow and Csáky assays, which detect catecholate and hydroxamate structures, respectively. Years later, the issue of Legionella siderophores was revisited using the CAS assay, which detects iron chelators independently of structure (Goldoni et al, 1991). CAS reactivity was detected in statically grown L. pneumophila cultures, suggesting the existence of a non-catecholate, non-hydroxamate siderophore. However, a later study determined that the CAS reactivity was due to the cysteine in the chemically defined growth medium (Liles and Cianciotto, 1996). When supernatants were derived from cultures generated with cysteine-free media, no siderophore activity was detected using the CAS-, Arnow-, or Csáky assays (Liles and Cianciotto, 1996; James et al, 1997). 1.2.2. The Discovery of Legiobactin The story of Legionella siderophores changed in 2000, when it was shown that L. pneumophila could excrete a high-affinity iron-chelator (Liles et al, 2000). Indeed, when grown with shaking at 37oC in a low-iron, chemically defined medium (CDM), L. pneumophila strains secrete a substance that is highly reactive in the CAS assay. Importantly, the siderophore activity is only seen when cultures are inoculated with bacteria that had been grown to log or early-stationary phase. Inocula derived from late-stationary phase cultures, despite growing in the CDM, do not elaborate CAS reactivity. L. pneumophila CAS reactivity was observed for virulent serogroup 1 strains 130b (also known as AA100 and Wadsworth), Philadelphia-1, and Oxford-4032E, as well as clinical and environmental isolates representing all nine of the other L. pneumophila serogroups tested (Liles et al, 2000). The nature of the CAS reactivity has been studied further in L. pneumophila strain 130b, and all data indicate that it represents a bona fide siderophore (Liles et al, 2000). First, the presence of CAS reactivity correlates with enhanced aerobic

2. Iron Assimilation and Type II Protein Secretion

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growth in an iron-deplete defined medium. Second, the chelating activity is subject to iron-repression; i.e., the addition of iron to the CDM decreases reactivity, whereas the removal of additional iron from the CDM cultures increases CAS reactivity. Third, the CAS reactivity is less than 1 kDa and is resistant to heat and proteases. Fourth, CAS reactions are rapid and intense. Fifth, CAS-positive supernatants facilitate the growth of wild-type legionellae in BCYE agar containing otherwise inhibitory concentrations of the iron chelator 2,2’-dipyridyl (DIP) (Allard et al, 2005). The iron-chelating activity in L. pneumophila supernatants is known as legiobactin (Liles et al, 2000). CAS-reactive supernatants remain negative in the Arnow, and Csáky assays, implying that legiobactin is not a typical catecholate or hydroxamate. In support of this notion, solvents used to extract common siderophores do not extract the Legionella siderophore (Liles et al, 2000). Legiobactin has been purified and its structure is nearly determined (K. A. Allard and N. P. Cianciotto, unpublished results). Representatives of most other Legionella species tested, including clinical and environmental isolates, also secrete CAS reactivity (Liles et al, 2000; Starkenburg et al, 2004). Although it cannot be concluded these species are producing legiobactin, their supernatants, like that of the L. pneumophila strains, are negative in the Arnow and Csáky assays. Some Legionella species do not display significant CAS reactivity (Starkenburg et al, 2004). However, these bacteria grow poorly in the CDM, because the medium is lacking an undefined growth factor other than iron. Thus, it is uncertain as to whether these legionellae lack a siderophore, since the absence of CAS reactivity could have been an indirect effect of minimal growth. 1.2.3. Genetics of Legiobactin Production The first gene to be examined as a possible promoter of legiobactin production was frgA, the ferric-regulated gene A identified in 1997 as an iron-regulated locus in strain 130b (Hickey and Cianciotto, 1997). Although FrgA is homologous with several hydroxamate synthetases, including enzymes involved in the biosynthesis of aerobactin in E. coli and Shigella species, alcaligin in Bordetella, and rhizobactin in Sinorhizobium (Hickey and Cianciotto, 1997), frgA mutants proved not to be defective for CAS reactivity (Liles et al, 2000). However, a L. pneumophila gene that is involved in legiobactin production has been recently uncovered. This gene, called lbtA for legiobactin gene A, encodes a product that is also homologous to multiple hydroxamate synthetases. Most importantly, several independently derived lbtA mutants have ca. 50% less CAS-reactive material when they are grown in iron-deplete CDM, indicating that LbtA is required for legiobactin production (Allard et al, 2005). The gene (lbtB) immediately downstream of lbtA is also required for full siderophore expression. Whereas LbtA is predicted to be involved in the biosynthesis of legiobactin, LbtB is likely involved in siderophore export, as it is a member of a family of transmembrane proteins associated with the export of small molecules such as siderophores. Several Fur boxes precede lbtA, suggesting that Fur mediates the iron regulation associated with legiobactin

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production. LbtA mutants show impaired growth in deferrated CDM that contains citrate, indicating that legiobactin facilitates extracellular growth in low-iron conditions. Southern blots show that lbtA is distributed among Legionella strains, a result compatible with the fact that many species produce CAS reactivity (Allard et al, 2005). 1.2.4. The Role of lbtA and frgA in Infection To start to gauge the role of siderophores in Legionella infection, the ability of the lbtA mutant to infect human U937 macrophages and Hartmannella vermiformis amoebae was determined. Although lbtA was expressed intracellularly, the mutant was unimpaired for infection of host cells, suggesting that LbtA and legiobactin are relevant but not required for intracellular infection (Allard et al, 2005). These data do not, however, indicate that siderophores are not needed intracellularly; the loss of legiobactin might be compensated for by another siderophore. Along those lines, frgA mutants are defective for growth in macrophages (Hickey and Cianciotto, 1997), raising the possibility that L. pneumophila encodes a siderophore that, unlike legiobactin, is necessary for optimal intracellular replication. Since frgA is absent from non-pneumophila strains (Hickey and Cianciotto, 1997), it is tempting to speculate that such a siderophore would be specific to L. pneumophila. 1.2.5. Summary and Current Models With the discovery of legiobactin and lbtA, we now, after years of disbelief, have strong evidence for the existence of a Legionella siderophore. It is probable that the novel influence of the bacterial inoculum on siderophore production is the main reason that legiobactin was not detected earlier. In retrospect, it is not surprising that L. pneumophila produces a siderophore, since many other aquatic bacteria produce this type of iron scavenger. Based upon the behavior of lbtA mutants, legiobactin is most critical for extracellular, rather than intracellular, growth. Thus, it is conceivable that the siderophore is vital for growth in aquatic environments such as biofilms. Although lbtA shares homology with enzymes involved in the biosynthesis of several well-studied hydroxamates, legiobactin may prove to be novel in structure, for several reasons. First, the observed CAS-reactivity is Csákyand Arnow-negative. Second, the L. pneumophila genome has not revealed additional genes (other than frgA) that would be predicted to promote the biosynthesis of a known hydroxamate. Although a biochemical activity associated with frgA has not been defined, the gene, based upon sequence data, is still a candidate siderophore gene. At present, the argument for frgA having a role in iron assimilation is that the gene is iron-regulated and required for optimal infection of macrophages. These data, coupled with the fact that LbtA mutants are not completely lacking CAS reactivity, suggests that L. pneumophila elaborates multiple siderophores. Whereas legiobactin is linked to extracellular growth, a second siderophore might be critical for intracellular infection. That L. pneumophila uses multiple siderophores in order to flourish in different niches is quite reasonable.


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1.3. Ferrous Iron Transport Since Fe2⫹ is present in the eukaryotic iron pool, presumably available to intracellular legionellae, the means by which L. pneumophila acquires and uses ferrous iron has been recently addressed. Toward that end, the feoB gene was identified and mutated in L. pneumophila strain 130b (Robey and Cianciotto, 2002). L. pneumophila FeoB shares 44% identity and 61% similarity with E. coli FeoB. Like its E. coli counterpart, Legionella FeoB has ten transmembrane domains, consistent with an inner membrane location. The gene’s promoter region has a putative Fur binding site, suggesting that L. pneumophila feoB is subject to iron regulation. feoB mutants exhibit decreased ferrous, but not ferric, radiolabeled iron uptake (Robey and Cianciotto, 2002). Growth of L. pneumophila in standard media is unaffected by the loss of FeoB. However, the feoB mutant has reduced growth on low-iron BCYE agar (Robey and Cianciotto, 2002). Compatible with its iron uptake defect, the mutant shows accelerated colonial growth on low-iron agar when plated around wells containing ferric but not ferrous iron salts. Additionally, the feoB mutant shows accelerated colonial growth when plated around wells containing legiobactin. The feoB mutant also has a growth defect in low-iron BYE broth (Robey and Cianciotto, 2002). This defect is exacerbated by the addition of DIP to the media, a result that can be reversed by additional supplementation with ferric chloride (Robey and Cianciotto, 2002). Together, these data indicate that L. pneumophila FeoB and Fe2⫹ transport are required for extracellular growth in low-iron conditions. Although aerobic conditions can result in a predominance of ferric iron, ferrous iron is present within Legionella cultures. FeoB might also transport Fe2⫹ generated in the periplasm, following reduction of acquired Fe3⫹ by a periplasmic ferric reductase (see below). The role of FeoB in intracellular infection has been assessed using U937 cells and H. vermiformis amoebae (Robey and Cianciotto, 2002). In the macrophage cell line, the feoB mutant exhibits a 10- to 15-fold reduced recovery compared to wildtype. Further evidence of the role of L. pneumophila FeoB in macrophage infection was seen using a cytopathicity assay. Whereas ⬎95% of the host cells are destroyed 72h after inoculation with wildtype bacteria, the viability of macrophages infected with FeoB-deficient bacteria does not decrease significantly over the assay period. A correlation between iron levels in macrophages and the growth defect observed in the feoB mutant has been obtained from infection assays using U937 cells that were depleted of iron by treatment with DIP. Indeed, the 15-fold growth defect seen with untreated cells increases to 105-fold in the presence of 50 ␮M DIP, respectively. When amoebal cultures were done in the presence of 50 ␮M DIP, the feoB mutant exhibited a 150-fold reduction in growth, relative to wildtype. Together, these results indicate that feoB and ferrous transport are important for the growth and survival of L. pneumophila in macrophages and amoebae (Robey and Cianciotto, 2002). The role of feoB in disease was examined using a competition assay and the A/J mouse model of pneumonia (Robey and Cianciotto, 2002). Bacteria were recovered from the

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lungs of mice following intratracheal inoculation with equal numbers of wildtype and mutant. At 3 days post-inoculation, the wildtype had outgrown the mutant by threefold, indicating that FeoB promotes in vivo growth. In summary, L. pneumophila FeoB is a ferrous iron transporter that is important for extracellular and intracellular growth. The infectivity defect of the feoB mutant indicates that ferrous iron is present and accessible in the L. pneumophila intracellular niche. That the feoB mutant is not completely defective for intracellular growth and only exhibits a modest virulence defect in mice implies that other uptake systems and/or iron sources can overcome decreased ferrous iron uptake. These data represent the first evidence for the importance of ferrous iron transport for intracellular replication by a human pathogen.

1.4. The L. pneumophila Iron Acquisition/Assimilation Locus Following a screen for mutants that are hypersensitive to the iron chelator EDDA and/or resistant to streptonigrin, the L. pneumophila iraAB (iron acquisition/assimilation) locus was identified (Pope et al, 1996). Importantly, insertions within iraA result in a severe lag in intracellular growth and a slowed rate of replication such that macrophage monolayers infected with iraA mutants yield 1000-fold fewer bacteria (Pope et al, 1996; Viswanathan et al, 2000). This intracellular growth defect is exacerbated by treatment of the host cells with desferrioxamine, confirming that the iraA mutants are defective for intracellular iron acquisition (Pope et al, 1996; Viswanathan et al, 2000). The iraA gene encodes a 272-aa protein that shows sequence similarity to methyltransferases that use Sadenosylmethionine as a donor. The iraB gene encodes a 501-aa protein that is highly similar to the PTR2 family of di- or tripeptide transporters and is predicted to be an inner membrane protein with 12 membrane spanning domains. A mutant containing an insertion in iraB consistently shows reduced growth in iron-depleted BYE broth (a phenotype eventually lost by iraA mutants) but does not have a defect in macrophages (Viswanathan et al, 2000). Thus, L. pneumophila iraA is critical for intracellular infection, although it is still not clear how IraA promotes infection or serves as a dispensable facilitator of iron assimilation. In contrast, iraB is most critical for growth under low-iron extracellular conditions. IraB may be involved in a novel pathway that imports iron-loaded peptides as an iron source. iraAB is conserved among strains of L. pneumophila but largely absent from other legionellae (Viswanathan et al, 2000).

1.5. Cytochrome C Maturation and Iron Assimilation Mutations within the cytochrome c maturation (ccm) locus greatly diminish the ability of L. pneumophila to grow on low-iron media; e.g., ccmB, ccmC, and ccmF mutants have a greatly reduced efficiency of plating on BCYE agar lacking added iron (Viswanathan et al, 2002; Naylor and Cianciotto, 2004). Ccm mutants are also dramatically impaired for replication in macrophages, amoebae, and the lungs of A/J mice (Viswanathan et al, 2002; Naylor and Cianciotto, 2004). The mutants’


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intracellular infectivity defects are exacerbated by treatment of the macrophages with desferrioxamine but ameliorated somewhat by the addition of iron, indicating that L. pneumophila ccm is required for optimal intracellular iron assimilation (Viswanathan et al, 2002; Naylor and Cianciotto, 2004). Bacterial Ccm systems are generally hypothesized to mediate the transfer of heme into the periplasm and then the attachment of heme to apocytochrome c (Cianciotto et al, 2005). Indeed, L. pneumophila ccm mutants lack cytochrome c (Viswanathan et al, 2002). However, the observations made with L. pneumophila are the fourth example of a linkage between ccm genes and bacterial iron assimilation, complementing studies with Paracoccus, Pseudomonas, and Rhizobium species (Cianciotto et al, 2005). The manner in which ccm promotes iron assimilation is unclear, although, in the case of the other bacteria, ccm mutants have changes in siderophore expression. Since the L. pneumophila ccm mutants produce normal levels of CAS reactivity (Viswanathan et al, 2002), it is possible that the Legionella ccm locus facilitates growth in low-iron conditions by a novel mechanism. The infectivity defects of the L. pneumophila ccm mutants indicate, for the first time, that ccm can be necessary for growth within an intracellular niche.

1.6. Legionella Ferric Reductases Legionella pneumophila encodes a cytoplasmic and a periplasmic ferric reductase (Johnson et al, 1991; Poch and Johnson, 1993; James et al, 1997). The purified periplasmic and cytoplasmic enzymes respectively use glutathione and NADPH as favored reductants (Poch and Johnson, 1993; James et al, 1997). Both enzymes are expressed within iron-limited and iron-replete cultures and within virulent and avirulent organisms (Poch and Johnson, 1993; James et al, 1997). Although ferric citrate can serve as a substrate for the purified reductases, it need not be the most physiologically relevant substrate.

1.7. Concluding Remarks Legionella pneumophila uses multiple pathways for iron acquisition. It may produce several siderophores; i.e., there is biochemical and genetic evidence for legiobactin, and genetic data for the existence of an hydroxamate-like iron chelator. Based upon observations with other bacteria, it is possible that Ccm proteins influence Legionella siderophores. Once internalized, ferrisiderophores may be acted upon by the periplasmic ferric reductase, yielding ferrous iron that would be transported across the inner membrane by FeoB. Alternatively, a yet-to-be described transporter might deliver the ferrisiderophore across the inner membrane, whereupon the cytoplasmic ferric reductase would generate ferrous iron. L. pneumophila environments contain significant amounts of ferrous iron, and thus a Fe2⫹-uptake pathway may also feed into FeoB. As yet another pathway, the L. pneumophila iraAB locus may encode an inner-membrane transporter that imports iron-loaded peptides. In addition to utilizing ferric and ferrous iron, L. pneumophila can bind and use hemin, in part through the action of the Hbp

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protein (O’Connell et al, 1996). The existence of multiple iron uptake systems in L. pneumophila is quite compatible with the fact that the bacterium resides within such a variety of environments.

2. Type II Protein Secretion 2.1. Type II Protein Secretion in Gram-Negative Bacteria In Gram-negative bacteria, type II protein secretion (T2S) is one of five protein secretion systems that permit the export of proteins from within the bacterial cell to the extracellular milieu and/or into target host cells. An analysis of the many sequenced genomes reveals that T2S genes are common but not universal within Gram-negatives (Cianciotto, 2005). Recent functional studies indicate that T2S can promote the virulence of human, animal, and plant pathogens as well as the physiology of various environmental bacteria (Cianciotto, 2005). T2S can be viewed as a two-step process (Filloux, 2004). In the first step, proteins destined for export are translocated across the inner membrane by the Sec or Tat pathway and then N-terminally cleaved by signal peptidase. In the second step, the proteins are transported from the periplasm to the exterior through the action of a complex of proteins specifically dedicated to T2S (i.e., the secreton), ultimately resulting in the passage of the proteins through an outer membrane pore (i.e., secretin). From work in several model organisms, such as Klebsiella, Pseudomonas, and Vibrio, there is now a relatively good understanding of the components of the T2S apparatus and the mechanism of the secretion process (Filloux, 2004). It is also clear that the T2S apparatus bears an evolutionary relationship to the type 4 pilus apparatus (Peabody et al, 2003). From the analysis of approximately a dozen different genera, the T2S can be viewed as consisting of 12 core components; i.e., the outer membrane secretin (T2S D), a cytoplasmic ATPase (T2S E), an inner (trans)membrane protein (T2S F), the major (T2S G) and minor (T2S H, I, J, K) pseudopilins, facilitators of the ATPase attachment to the inner membrane that appear, along with T2S F, to form an inner membrane platform (T2S L, M), the pre-pseudopilin peptidase/methyltransferase (T2S O) that also acts upon type 4 prepilin, and a protein that may be involved in substrate recognition and/or secretin interactions (T2S C) (Peabody et al, 2003; Filloux, 2004). After translocation across the inner membrane, a protein destined for export is cleaved and its nascent N-terminus methylated by the pre-pseudopilin peptidase (also commonly known as PilD). The processed protein is then recognized for entry into the T2S apparatus by a mechanism that is not understood but likely involves tertiary structural determinants. The proteins ultimately exit the cell through the secretin pore in an energydependent process, with the pseudopilins forming a pilus-like structure that appears like a piston to push the proteins out through the secretin.


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2.2. Discovery of Type II Secretion in Legionella pneumophila The first indication that L. pneumophila has a T2S system was a 1998 report of a pilBCD operon, encoding the pre-pseudopilin peptidase T2S O (PilD), along with PilB and PilC proteins, which are strictly dedicated to type 4 pilus biogenesis (Liles et al, 1998). A subsequent report then showed that a mutation in the pilD gene of L. pneumophila serogroup-1 strain 130b reduced protein secretion as evidenced by the loss of proteins in mutant culture supernatants visualized by 1-dimensional SDS-PAGE (Liles et al, 1999). A later study in L. pneumophila strain Philadelphia-1 reported the presence of a locus, lspFGHIJK, predicted to encode homologs of the T2S FGHIJK proteins (Hales and Shuman, 1999). As was the case for mutation of pilD, mutation of lspGH resulted in loss of protein secretion as measured by 1-dimensional SDS-PAGE. Two further studies in L. pneumophila strain 130b reported the presence of genes encoding homologs of T2S DE (lspDE), C (lspC), and LM (lspLM) (Rossier and Cianciotto, 2001; Rossier et al, 2004). Together, these studies indicated that L. pneumophila has a complete and functional type II protein secretion system. This was confirmed when, in 2004, the complete genomes of L. pneumophila serogroup 1 strains Philadelphia 1, Paris, and Lens were published (Cazalet et al, 2004; Chien et al, 2004; Cianciotto, 2005). Southern hybridization analysis has demonstrated that T2S genes are also in other serogroups of L. pneumophila and other species of Legionella (Rossier et al, 2004). Genome sequencing of several strains has also revealed that L. pneumophila encodes Sec and Tat systems for translocating proteins across the inner membrane into the periplasm (Cazalet et al, 2004; Chien et al, 2004; De Buck et al, 2004; Rossier and Cianciotto, 2005). That L. pneumophila sec is essential for growth and necessary for protein secretion is unquestioned. Yet, recent studies have confirmed that the genes encoding the twin-arginine translocation system are expressed during L. pneumophila extra- and intracellular growth (De Buck et al, 2004) and are functional as judged by the negative impact that their mutation has on cytochrome c-dependent respiration and the processing of the cytochrome c reductase PetA (Rossier and Cianciotto, 2005). As expected, L. pneumophila also has and expresses the type I signal peptidase (LepB), which cleaves the N-terminus from proteins translocated across the inner membrane (Lammertyn et al, 2004). Thus, not only does L. pneumophila possess the full set of T2S-specific genes, but it also has the machinery for delivering proteins into the periplasm in order that they might then enter the secretion apparatus. A variety of T2S-specific mutants have been constructed and characterized. All mutants replicate normally within standard BYE broth incubated at 37°C (Rossier and Cianciotto, 2001; Rossier et al, 2004). They also have a typical efficiency of plating on standard BCYE agar at 37°C. However, the mutants do display a slightly altered colony morphology on the BCYE agar (Rossier and Cianciotto, 2001; Rossier et al, 2004).

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2.3. Factors Secreted by the Type II System of L. pneumophila Presently, at least 11 enzymes have been determined to be secreted via the L. pneumophila T2S system (Rossier et al, 2004; Banerji et al, 2005; Cianciotto, 2005). This conclusion is based upon the loss of enzymatic activities from the culture supernatants of lspDE, lspF, lspG, lspGH, or pilD mutants grown in standard BYE broth at 37°C (Hales and Shuman, 1999; Liles et al, 1999; Aragon et al, 2000; Rossier and Cianciotto, 2001; Rossier et al, 2004). The T2S-dependent proteins/activities include tartrate-sensitive and tartrate-resistant acid phosphatases, phospholipase A, phospholipase(s) C, lysophospholipase A, glycerophospholipid:cholesterol acyltransferase (GCAT), mono-, di-, and triacylglycerol lipases, ribonuclease, and protease (Hales and Shuman, 1999; Liles et al, 1999; Aragon et al, 2000; Aragon et al, 2001; Flieger et al, 2001b; Rossier and Cianciotto, 2001; Aragon et al, 2002; Flieger et al, 2002; Rossier et al, 2004; Banerji et al, 2005). Because mutants specifically lacking the type 4 pilus are not defective for any of these activities, the altered secretion of the pilD mutants is due to the loss of T2S (Rossier and Cianciotto, 2001; Rossier et al, 2004). Several of the secreted enzymatic activities have now been identified in other species of Legionella (Flieger et al, 2001a; Rossier et al, 2004). In some cases, the structural genes (proteins) encoding T2S-dependent secreted activities have been identified. These include map (Map) for the tartrate-sensitive acid phosphatase (Aragon et al, 2001), plcA (PlcA) for phospholipase C activity (Aragon et al, 2002), plaA (PlaA) for the lysophospholipase A (Flieger et al, 2002), plaC (PlaC) for GCAT (Banerji et al, 2005), lipA (LipA) and lipB (LipB) for mono- and triacylglycerol lipases (Aragon et al, 2002), and proA/msp (ProA/Msp) the zinc metalloprotease (Hales and Shuman, 1999; Liles et al, 1999). Notably, mutations in any one of these genes do not completely abolish the corresponding activity, suggesting that L. pneumophila has more than one secreted phosphatase, phospholipase C, lysophospholipase A, lipase, and protease/peptidase and that the Legionella T2S system mediates the secretion of more than 11 proteins. Indeed, 2-dimensional SDS-PAGE analysis of wild type strain 130b and its lsp mutant derivatives indicate that more than 30 L. pneumophila proteins are T2S-dependent (DebRoy, S., and N.P. Cianciotto, unpublished results). Given the completion of the L. pneumophila genome, mass spectrometric analysis of protein spots extracted from 2-D gels is now allowing a direct determination of those secreted proteins that are linked to the T2S system. In addition to confirming the T2S-dependency of previously described proteins (e.g., ProA/Msp, Map, PlaA), this type of analysis is defining for the first time the genetic basis for other previously described enzymatic activities (e.g., ribonuclease) as well as uncovering new secreted enzymatic activities (e.g., chitinase, aminopeptidase) (DebRoy, S., and N.P. Cianciotto, unpublished results). The analysis of supernatants from proA/msp mutants is demonstrating that some secreted proteins are subject to cleavage and perhaps activation by the T2S-dependent ProA/Msp metalloprotease (Flieger et al,


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2002; Banerji et al, 2005). In some instances, the T2S-dependent effectors of L. pneumophila are closely related to secreted proteins of other bacteria; e.g., ProA/Msp is homologous with well-characterized proteases from a variety of Gram-negative organisms, and PlcA is highly related to a new type of phospholipase C found in Pseudomonas (Aragon et al, 2002). However, in the case of the Map, the L. pneumophila protein is most related to eukaryotic acid phosphatases (Aragon et al, 2001); the fact that L. pneumophila encodes a number of proteins that bear their greatest homology with eukaryotic proteins has more recently emerged from the annotation of the completed genome (Cazalet et al, 2004; Chien et al, 2004). By virtue of a twin-arginine in its signal peptide, PlcA was predicted to be a Tat substrate; indeed mutations in tatB diminish secreted phospholipase C activity (Rossier and Cianciotto, 2005).

2.4. Role of Type II Secretion in Pathogenesis Legionella pneumophila T2S mutants (i.e., strain 130b derivatives containing an insertion mutation in lspDE, lspF, lspG, lspK, or pilD) display a reduced ability to infect human macrophages, including the U937 cell line and peripheral blood mononuclear cell-derived macrophages (Liles et al, 1999; Polesky et al, 2001; Rossier and Cianciotto, 2001; Rossier et al, 2004). In the U937 cells, the lsp mutants were modestly defective, showing a tenfold reduced recoverability at 48 hours post-inoculation (Polesky et al, 2001; Rossier and Cianciotto, 2001; Rossier et al, 2004). In contrast, an lsp mutant appeared incapable of replication within macrophages derived from peripheral blood cells, indicating that the magnitude of the mutant phenotype is influenced by the nature of the target host cell. Indeed, when tested in the human HL60 cell line, an lspGH mutant behaved in a manner like wild type (Hales and Shuman, 1999). The reduced infectivity of pilD and lsp mutants was complemented by reintroduction of the corresponding gene (Rossier et al, 2004). That L. pneumophila mutants specifically lacking type 4 pili are not defective for macrophage infection indicates that the altered infectivity of the pilD mutant is due to the loss of T2S (Rossier et al, 2004). The T2S mutants do not appear to be defective for entry but rather lack the capacity to optimally replicate (Rossier et al, 2004). They are not impaired for the induction of apoptosis or a pore-forming activity linked to egress from host cells (Molmeret et al, 2002; Zink et al, 2002). Together, these data indicate that T2S promotes L. pneumophila infection of macrophages. Recently, it has been shown that lsp mutants are defective in an animal model of Legionnaires’ disease pneumonia (Rossier et al, 2004). More specifically, upon intratracheal inoculation into A/J mice, lspDE, lspF, and pilD mutants, but not pilus mutants, exhibit a reduced ability to grow in the mammalian lung, as measured by competition assays. Additionally, the lspF was defective in an in vivo growth kinetics assay; i.e., whereas wild type bacteria increased at least tenfold in the A/J mouse lung, the T2S mutant failed to show any evidence of replication. Examination of infected mouse sera revealed that type-II secreted proteins are expressed in vivo. Thus, the L. pneumophila type II secretion is an

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important virulence factor. The reduced survival of the lsp mutants in the A/J mouse lung is likely due, at least in part, to diminished growth in alveolar macrophages. However, that lspF mutant CFU do not increase in the mouse lungs, whereas they do, albeit not optimally, in vitro, raises the possibility that the lsp mutants are also defective for extracellular processes that are operative in the lungs, such as combating other aspects of the innate immune response. The various mutants that lack particular T2S effectors have also been tested for alterations in intracellular infection. However, map, plcA, plaA, plaC, lipA, lipB, and proA/msp mutants all grow normally within macrophages, indicating that the tartrate-sensitive acid phosphatase, PlcA phospholipase C, PlaA lysophospholipase A, PlaC GCAT, LipA and LipB lipases, and the zinc metalloprotease are not required for intracellular infection (Aragon et al, 2001, 2002; Flieger et al, 2002; Banerji et al, 2005). These results suggest that the T2S system secretes a yet-tobe-defined factor that is required for infection. Alternatively, there may be redundancy in the effectors, such that the loss of one enzyme can be compensated with a similar type of enzyme. Clearly, more work is needed in order to fully explain the role of T2S in pathogenesis. However, these various studies do represent the first documentation of a role for T2S in intracellular infection and in an animal model of disease.

2.5. Role of Type II Secretion in Environmental Survival Legionella pneumophila lsp and pilD mutants are severely defective for intracellular growth in fresh water amoebae, including H. vermiformis and Acanthamoeba castellanii (Hales and Shuman, 1999; Liles et al, 1999; Polesky et al, 2001; Rossier and Cianciotto, 2001; Rossier et al, 2004). Indeed, the mutants show little, if any, evidence of replication within the protozoan hosts. The reduced infectivity of the mutants is complemented by reintroduction of the corresponding intact gene, and thus these data clearly demonstrate that T2S is required for amoebal infection. As was the case for macrophage infection, studies using mutants lacking particular secreted enzymes have failed to identify the T2S effector(s) that is critical for protozoan infection. Nonetheless, given the absolute importance of protozoan infection for L. pneumophila survival in aquatic habitats, these data establish T2S as a highly significant promoter of L. pneumophila persistence in environmental niches. That T2S promotes protozoa infection may increase its relevance for pathogenesis, since infected protozoa have been hypothesized to be part of the infective dose (Brieland et al, 1996; Cirillo et al, 1999). Following upon the demonstration that pilD transcript levels are greater in legionellae grown at 30 vs. 37°C (Liles et al, 1998), it was shown, using a pilD::lacZ fusion strain, that pilD transcriptional initiation increases progressively as L. pneumophila strain 130b is grown at 30, 25, and 17°C (Söderberg et al, 2004). Legionella pilD mutants also had a dramatically reduced ability to grow in BYE broth and to form colonies on BCYE agar at the lower temperatures. Whereas strains specifically lacking pili were not defective for low-temperature growth, lsp mutants were greatly impaired for colony formation at 25, 17, and


45

12°C (Söderberg et al, 2004). Indeed, the lsp mutants were completely unable to grow at 12°C. The growth defects of the pilD and lsp mutants on the yeast extract media was complemented by reintroduction of the corresponding intact gene. In a series of experiments that more closely mimic aquatic habitats, an lsp mutant displayed reduced survivability in tap water incubated at 25, 17, 12, and 4°C (Söderberg, M.A., and N.P. Cianciotto, unpublished results). Together, these data indicate that T2S promotes the growth of L. pneumophila at low temperatures. Interestingly, the lsp mutants displayed improved growth at 25°C when plated next to a streak of wild type but not mutant bacteria, implying that a secreted, diffusible factor promotes low-temperature growth (Söderberg et al, 2004). Indeed, growth stimulation could be seen using filter-sterilized supernatants from wild type. Mutants lacking the known acid phosphatase, lipases, phospholipase C, lysophospholipase A, or protease grow normally at 25°C, suggesting the existence of a critical, yet-to-be-defined exoproteins(s) (Söderberg et al, 2004). Recent 2-D SDS-PAGE comparisons of supernatants from cultures grown at different temperatures reveal proteins that are only expressed or hyperexpressed at low temperatures (Söderberg, M.A., and N.P. Cianciotto, unpublished results). In summary, these data document, for the first time, that L. pneumophila replicates at temperatures below 20°C and that a bacterial T2S system facilitates growth at low temperatures.

2.6. Concluding Comments The large amount of data obtained from studies with macrophages, experimental animals, protozoa, and low-temperature conditions show how the Legionella T2S system promotes bacterial growth both in a mammalian host and in environmental niches. Future work will assess whether the same or distinct T2S effectors are critical for growth in the disparate niches. Nonetheless, L. pneumophila Lsp already represents the best-characterized T2S system in terms of understanding the importance of the system in a bacterium’s physiology, pathogenesis, and ecology (Cianciotto, 2005).

Acknowledgments. Studies in the author’s laboratory are supported by NIH grants AI34937 and AI43987.

References Allard, K.A., Viswanathan, V.K., and Cianciotto, N.P. (2005) Genes lbtA and lbtB are required for production of the Legionella pneumophila siderophore legiobactin. J. Bacteriol. submitted for publication. Aragon, V., Kurtz, S., Flieger, A., Neumeister, B., and Cianciotto, N.P. (2000) Secreted enzymatic activities of wild-type and pilD-deficient Legionella pneumophila. Infect. Immun. 68: 1855–1863. Aragon, V., Kurtz, S., and Cianciotto, N.P. (2001) Legionella pneumophila major acid phosphatase and its role in intracellular infection. Infect. Immun. 69: 177–185.

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Aragon, V., Rossier, O., and Cianciotto, N.P. (2002) Legionella pneumophila genes that encode lipase and phospholipase C activities. Microbiol. 148: 2223–2231. Banerji, S., Bewersdorff, M., Hermes, B., Cianciotto, N.P., and Flieger, A. (2005) Characterization of the major secreted zinc metalloprotease-dependent glycerophospholipid:cholesterol acyltransferase, PlaC, of Legionella pneumophila. Infect. Immun. 73: 2899–2909. Brieland, J., McClain, M., Heath, L., Chrisp, C., Huffnagle, G., LeGendre, M., Hurley, M., Fantone, J., and Engleberg, C. (1996) Coinoculation with Hartmannella vermiformis enhances replicative Legionella pneumophila lung infection in a murine model of Legionnaires’ disease. Infect. Immun. 64: 2449–2456. Byrd, T.F., and Horwitz, M.A. (2000) Aberrantly low transferrin receptor expression on human monocytes is associated with nonpermissiveness for Legionella pneumophila growth. J. Infect. Dis. 181: 1394–1400. Cazalet, C., Rusniok, C., Bruggemann, H., Zidane, N., Magnier, A., Ma, L., Tichit, M., Jarraud, S., Bouchier, C., Vandenesch, F., Kunst, F., Etienne, J., Glaser, P., and Buchrieser, C. (2004) Evidence in the Legionella pneumophila genome for exploitation of host cell functions and high genome plasticity. Nat. Genet. 36: 1165–1173. Chien, M., Morozova, I., Shi, S., Sheng, H., Chen, J., Gomez, S.M., Asamani, G., Hill, K., Nuara, J., Feder, M., Rineer, J., Greenberg, J.J., Steshenko, V., Park, S.H., Zhao, B., Teplitskaya, E., Edwards, J.R., Pampou, S., Georghiou, A., Chou, I.C., Iannuccilli, W., Ulz, M.E., Kim, D.H., Geringer-Sameth, A., Goldsberry, C., Morozov, P., Fischer, S.G., Segal, G., Qu, X., Rzhetsky, A., Zhang, P., Cayanis, E., De Jong, P.J., Ju, J., Kalachikov, S., Shuman, H.A., and Russo, J.J. (2004) The genomic sequence of the accidental pathogen Legionella pneumophila. Science 305: 1966–1968. Cianciotto, N.P. (2005) Type II secretion: A protein secretion system for all seasons. Trends Microbiol. 13:581–588. Cianciotto, N.P., Cornelis, P., and Baysse, C. (2005) Impact of the bacterial type I cytochrome c maturation system on different biological processes. Mol. Microbiol. 56: 1408–1415. Cirillo, J.D., Cirillo, S.L.G., Yan, L., Bermudez, L.E., Falkow, S., and Tompkins, L.S. (1999) Intracellular growth in Acanthamoeba castellani affects monocyte entry mechansism and enhances virulence of Legionella pneumophila. Infect. Immun. 67: 4427–4434. De Buck, E., Lebeau, I., Maes, L., Geukens, N., Meyen, E., Van Mellaert, L., Anne, J., and Lammertyn, E. (2004) A putative twin-arginine translocation pathway in Legionella pneumophila. Biochem. Biophys. Res. Commun. 317: 654–661. Filloux, A. (2004) The underlying mechanisms of type II protein secretion. Biochim. Biophys. Acta-Mol. Cell. Res. 1694: 163–179. Flieger, A., Gong, S., Faigle, M., Northoff, H., and Neumeister, B. (2001a) In vitro secretion kinetics of proteins from Legionella pneumophila in comparison to proteins from non-pneumophila species. Microbiol. 147: 3127–3134. Flieger, A., Gong, S., Faigle, M., Stevanovic, S., Cianciotto, N.P., and Neumeister, B. (2001b) Novel lysophospholipase A secreted by Legionella pneumophila. J. Bacteriol. 183: 2121–2124. Flieger, A., Neumeister, B., and Cianciotto, N.P. (2002) Characterization of the gene encoding the major secreted lysophospholipase A of Legionella pneumophilaand its role in detoxification of lysophosphatidylcholine. Infect. Immun. 70: 6094–6106. Gebran, S.J., Newton, C., Yamamoto, Y., Widen, R., Klein, T.W., and Friedman, H. (1994) Macrophage permissiveness for Legionella pneumophila growth modulated by iron. Infect. Immun. 62: 564–568.


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Goldoni, P., Visca, P., Pastoris, M.C., Valenti, P., and Orsi, N. (1991) Growth of Legionella spp. under conditions of iron restriction. J. Med. Microbiol. 34: 113–118. Hales, L.M., and Shuman, H.A. (1999) Legionella pneumophila contains a type II general secretion pathway required for growth in amoebae as well as for secretion of the Msp protease. Infect. Immun. 67: 3662–3666. Hickey, E.K., and Cianciotto, N.P. (1994) Cloning and sequencing of the Legionella pneumophila fur gene. Gene 143: 117–121. Hickey, E.K., and Cianciotto, N.P. (1997) An iron- and fur-repressed Legionella pneumophila gene that promotes intracellular infection and encodes a protein with similarity to the Escherichia coli aerobactin synthetases. Infect. Immun. 65: 133–143. James, B.W., Mauchline, W.S., Fitzgeorge, R.B., Dennis, P.J., and Keevil, C.W. (1995) Influence of iron-limited continuous culture on physiology and virulence of Legionella pneumophila. Infect. Immun. 63: 4224–4230. James, B.W., Mauchline, W.S., Dennis, P.J., and Keevil, C.W. (1997) A study of iron acquisition mechanisms of Legionella pneumophila grown in chemostat culture. Curr. Microbiol. 34: 238–243. Johnson, W., Varner, L., and Poch, M. (1991) Acquisition of iron by Legionella pneumophila: role of iron reductase. Infect. Immun. 59: 2376–2381. Lammertyn, E., Van Mellaert, L., Meyen, E., Lebeau, I., De Buck, E., Anne, J., and Geukens, N. (2004) Molecular and functional characterization of type I signal peptidase from Legionella pneumophila. Microbiol. 150: 1475–1483. Liles, M.R., and Cianciotto, N.P. (1996) Absence of siderophore-like activity in Legionella pneumophila supernatants. Infect. Immun. 64: 1873–1875. Liles, M.R., Viswanathan, V.K., and Cianciotto, N.P. (1998) Identification and temperature regulation of Legionella pneumophila genes involved in type IV pilus biogenesis and type II protein secretion. Infect. Immun. 66: 1776–1782. Liles, M.R., Edelstein, P.H., and Cianciotto, N.P. (1999) The prepilin peptidase is required for protein secretion by and the virulence of the intracellular pathogen Legionella pneumophila. Mol. Microbiol. 31: 959–970. Liles, M.R., Aber Scheel, T., and Cianciotto, N.P. (2000) Discovery of a nonclassical siderophore, legiobactin, produced by strains of Legionella pneumophila. J. Bacteriol. 182: 749–757. Mengaud, J.M., and Horwitz, M.A. (1993) The major iron-containing protein of Legionella pneumophila is an aconitase homologous with the human iron-responsive element-binding protein. J. Bacteriol. 175: 5666–5676. Molmeret, M., Alli, O.A., Zink, S., Flieger, A., Cianciotto, N.P., and Kwaik, Y.A. (2002) icmT is essential for pore formation-mediated egress of Legionella pneumophila from mammalian and protozoan cells. Infect. Immun. 70: 69–78. Naylor, J., and Cianciotto, N.P. (2004) Cytochrome c maturation proteins are critical for in vivo growth of Legionella pneumophila. FEMS Microbiol. Lett. 241: 249–256. O’Connell, W.A., Hickey, E.K., and Cianciotto, N.P. (1996) A Legionella pneumophila gene that promotes hemin binding. Infect. Immun. 64: 842–848. Peabody, C.R., Chung, Y.J., Yen, M.R., Vidal-Ingigliardi, D., Pugsley, A.P., and Saier, M.H., Jr. (2003) Type II protein secretion and its relationship to bacterial type IV pili and archaeal flagella. Microbiology 149: 3051–3072. Poch, M.T., and Johnson, W. (1993) Ferric reductases of Legionella pneumophila. Biometals 6: 107–114. Polesky, A.H., Ross, J.T., Falkow, S., and Tompkins, L.S. (2001) Identification of Legionella pneumophila genes important for infection of amoebas by signature-tagged mutagenesis. Infect. Immun. 69: 977–987.

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Pope, C.D., O’Connell, W., and Cianciotto, N.P. (1996) Legionella pneumophila mutants that are defective for iron acquisition and assimilation and intracellular infection. Infect. Immun. 64: 629–636. Reeves, M.W., Pine, L., Hutner, S.H., George, J.R., and Harrell, W.K. (1981) Metal requirements of Legionella pneumophila. J. Clin. Microbiol. 13: 688–695. Reeves, M.W., Pine, L., Neilands, J.B., and Balows, A. (1983) Absence of siderophore activity in Legionella species grown in iron-deficient media. J. Bacteriol. 154: 324–329. Robey, M., and Cianciotto, N.P. (2002) Legionella pneumophila feoAB promotes ferrous iron uptake and intracellular infection. Infect. Immun. 70: 5659–5669. Rossier, O., and Cianciotto, N.P. (2001) Type II protein secretion is a subset of the PilDdependent processes that facilitate intracellular infection by Legionella pneumophila. Infect. Immun. 69: 2092–2098. Rossier, O., and Cianciotto, N.P. (2005) The Legionella pneumophila tatB gene facilitates secretion of phospholipase C, growth under iron-limiting conditions, and intracellular infection. Infect. Immun. 73: 2020–2032. Rossier, O., Starkenburg, S., and Cianciotto, N.P. (2004) Legionella pneumophila type II protein secretion promotes virulence in the A/J mouse model of Legionnaires’ disease pneumonia. Infect. Immun. 72: 310–321. Söderberg, M.A., Rossier, O., and Cianciotto, N.P. (2004) The type II protein secretion system of Legionella pneumophila promotes growth at low temperatures. J. Bacteriol. 186: 3712–3720. Starkenburg, S.R., Casey, J.M., and Cianciotto, N.P. (2004) Siderophore activity among members of the Legionella genus. Curr. Microbiol. 49: 203–207. Viswanathan, V.K., Edelstein, P.H., Pope, C.D., and Cianciotto, N.P. (2000) The Legionella pneumophila iraAB locus is required for iron assimilation, intracellular infection, and virulence. Infect. Immun. 68: 1069–1079. Viswanathan, V.K., Kurtz, S., Pedersen, L.L., Abu-Kwaik, Y., Krcmarik, K., Mody, S., and Cianciotto, N.P. (2002) The cytochrome c maturation locus of Legionella pneumophila promotes iron assimilation and intracellular infection and contains a strain-specific insertion sequence element. Infect. Immun. 70: 1842–1852. Zink, S.D., Pedersen, L., Cianciotto, N.P., and Abu-Kwaik, Y. (2002) The Dot/Icm type IV secretion system of Legionella pneumophila is essential for the induction of apoptosis in human macrophages. Infect. Immun. 70: 1657–1663.

3 The Dot/Icm Type IVB Secretion System Jason J. LeBlanc and Joseph P. Vogel

1. Introduction As a fundamental strategy for virulence, a large number of pathogens possess specialized secretion systems that are used to deliver effector proteins into host cells, thus modulating host cell function. In Gram-negative bacteria, these specialized secretion systems include type III secretion systems (T3SSs) (Hueck, 1998) and type IV secretion systems (T4SSs) (Backert and Meyer, 2006; Christie and Vogel, 2000; Christie et al., 2005; Sexton and Vogel, 2002). T3SS components share similarities to flagellar biosynthetic proteins, whereas T4SSs are believed to be ancestrally related to bacterial conjugation systems (Sexton and Vogel, 2002; Backert and Meyer, 2006). Both T3SSs and T4SSs are multicomponent membrane complexes that can act as molecular syringes to directly inject bacterial proteins into target cells (Sexton and Vogel, 2002; Nagai and Roy, 2003; Backert and Meyer, 2006). T3SSs are present in many pathogens including a variety of Pseudomonas, Salmonella, Shigella, and Yersinia species (Hueck, 1998). T4SSs play equally important roles for the virulence of several pathogens including Brucella, Bordetella, Helicobacter pylori, and Legionella pneumophila (Backert and Meyer, 2006). T4SSs include both classical plasmid transfer systems and adapted conjugation systems used by pathogens (Christie and Vogel, 2000; Sexton and Vogel, 2002). The paradigm T4SS is generally considered to be the plasmid-encoded VirB system of Agrobacterium tumefaciens, which delivers both DNA and protein into plant cells (reviewed in Christie et al., 2005 and Christie and Vogel, 2000). Two additional well-characterized adapted conjugation systems include the Ptldependent secretion of pertussis toxin by Bordetella pertussis and the secretion of CagA by Helicobacter pylori (Backert and Meyer, 2006). T4SS-dependent virulence strategies are also thought to be used by intracellular pathogens such as Brucella spp. and Coxiella burnetii to generate a replication-permissive niche in host cells (Boschroli et al., 2002; Celli and Gorvel, 2004; Saver et al., 2005; Segal et al., 2005; Sexton and Vogel, 2002). The L. pneumophila T4SS, the focus of this chapter, is required for the intracellular survival and replication of this

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Jason J. LeBlanc and Joseph P. Vogel

bacterium. Though the global architecture of the L. pneumophila T4SS has not yet been fully elucidated, the following sections will describe several key components of the macromolecular membrane complex, as well as several effector proteins that may be involved in the generation of a suitable niche for the intracellular growth of this organism.

2. Dot/Icm Type IV Secretion System At least two different T4SS have been identified in a number of L. pneumophila strains. The first T4SS, a type IVA secretion system, was designated Lvh for “Legionella Vir homolog” due to its similarities to the virB operon of A. tumefaciens. However, this system was shown to be dispensable for intracellular replication of L. pneumophila and is not present in several virulent strains (Christie and Vogel, 2000; Segal et al., 1999). The second T4SS, a type IVB secretion system, was named the Dot/Icm system and shares extensive similarity to the Tra/Trb components of the conjugal transfer apparatus of the IncI plasmids R64 and ColIb-P9 (Berger and Isberg, 1993; Christie and Vogel, 2000; Marra et al., 1992; Segal and Shuman, 1998; Sexton and Vogel, 2002; Vogel et al., 1998). The L. pneumophila type IVB secretion system is encoded by 26 genes designated dot for “defect in organelle trafficking” or icm for “intracellular multiplication” and is found on two loci in the L. pneumophila genome termed region I and II (Sexton and Vogel, 2002). The simultaneous discovery of the dot/icm genes by two laboratories (Berger and Isberg, 1993; Marra et al., 1992) resulted in some components having dual Dot and Icm designations (Table 3.1) (Sexton and Vogel, 2002). Since its discovery, the Dot/Icm T4SS has become the most extensively studied virulence factor associated with L. pneumophila pathogenesis. In contrast to the Lvh T4SS, the Dot/Icm T4SS is absolutely required for the virulence of L. pneumophila. Unlike wild-type L. pneumophila, phagosomes containing dot/icm mutants fail to evade the endocytic pathway and quickly acquire endosomal markers such as the small GTPase Rab5, the vacuolar H⫹-ATPase (V-ATPase), and the lysosomal-associated membrane protein (LAMP)-1 (Coers et al., 1999; Lu and Clarke, 2005; Swanson and Hammer, 2000). L. pneumophila is thought to avoid endocytic degradation by reprogramming host trafficking via the action of Dot/Icmtranslocated effector proteins, thus generating a safe niche termed the “replicative phagosome.” Dot/Icm-dependent alteration of the host endocytic pathway is a rapid event and occurs within minutes of bacterial internalization (Roy et al., 1998; Wiater et al., 1998). Unlike effectors injected in the host by the Salmonella T3SSs (Uchiya et al., 1999), Dot/Icm-dependent secretion does not result in a global effect on phagosome formation in the host cell (Coers et al., 1999; Horwitz and Maxfield, 1984). This result was based partially on the observations that phagosomes harboring Saccharomyces cerevisiae enter the endocytic pathway in a normal

3. The Dot/Icm Type IVB Secretion System

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TABLE 3.1. Characteristics of the Legionella pneumophila Dot/Icm proteins. Proteina DotA

#aab 1048

Sizec 113

IncId TraY

DotB DotC DotD DotE/IcmC

377 303 163 194

42 34 18 20

TraJ TraI TraH TraQ

DotF/IcmG DotG//IcmE

269 1048

30 108

TraP TraO

DotH/IcmK

360

39

TraN

Peri/OM

DotI/IcmL DotJ/IcmM DotK/IcmN DotL/IcmO

212 94 189 783

23 11 21 87

TraM

IM IMh OM IM

DotM/IcmP DotN/IcmJ DotO/IcmB DotP/IcmD DotU DotV IcmF IcmQ IcmR IcmS IcmT IcmV IcmW IcmX

376 208 1009 132 261 180 973 191 120 114 86 151 151 466

43 24 112 14 30 20 111 22 13 13 10 18 17 51

TrbA TraT TraU TraR

TraL TrbC

TraK TraX TraW

Informatione Polytopic membrane protein VirB11-like Lipoprotein Lipoprotein Polytopic membrane protein VirB10-like

Lipoprotein VirD4-like/ Essential gene Essential gene Essential gene

Locationf IM Cyto OM OM IMh IM IM

IM Cyto/IM IM IM Accessory factor IM DotE homology IMh Accessory factor IM Pore forming molecule Cyto Chaperone for IcmQ Cyto Cyto IMh IM Cyto Peri

Functiong

ATPase

IM receptor Energy transducer Possible OM pore

Energy transducer

Chaperone Chaperone Pore? Chaperone Type IV adaptor

Type IV adaptor

a

Proteins are listed with the corresponding Dot or Icm designation. Number of amino acids. c Predicted size in kilo-Daltons. d Name of the IncI plasmid homolog. e Motifs or homology to the A. tumefaciens type IV secretion system. f Localization of the protein as determined experimentally (cyto is cytoplasm, IM is inner membrane, peri is periplasm, and OM is outer membrane). g Predicted function of the protein. h Localization of the protein by a variety of computer algorithms. b

manner in macrophages pre-infected with L. pneumophila (Coers et al., 1999). The lack of a global effect on trafficking by Legionella is likely due to retention of Dot/Icm-effectors on the cytoplasmic face of the Legionella-containing vacuole (LCV) (Bardill et al., 2005; Campodonico et al., 2005; Conover et al., 2003; Luo and Isberg, 2004; Nagai et al., 2002), thus allowing a more stealthy, ninja-like non-disruptive invasion of phagocytic cells.

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2.1. Structural Components The Dot/Icm system is a multiprotein structure that forms a channel spanning the bacterial inner and outer membranes (Coers et al., 1999). Although many of the Dot/Icm components have not been extensively characterized, a number of them have now been examined revealing their localization and putative roles in the formation/function of the secretion apparatus. The first to be examined, DotA, is a polytopic inner membrane protein that may act as a scaffolding protein (Roy and Isberg, 1997). DotB is a cytoplasmic, hexameric protein that associates with the inner membrane and possesses ATPase activity. DotB ATPase activity is crucial for the function of the Dot/Icm complex since mutations in the Walker A box motif (ATP-binding domain) result in a protein that cannot hydrolyze ATP or complement a dotB null mutant (Sexton et al., 2004b, 2005; Vogel et al., 1996, 1998). Current models suggest that DotB is an ATPase that acts as a molecular pump driving export of substrates through the Dot/Icm complex via hydrolysis of ATP while providing substrate specificity to the apparatus (Sexton et al., 2004b). IcmX was shown to be periplasmic protein, although its role in the apparatus remains unclear (Matthews and Roy, 2000). Interestingly, IcmX homologues have not been observed in other T4SSs. Five proteins, DotF/DotG/DotH/ DotC/DotD appear to form the core transmembrane complex for the L. pneumophila T4SS (Vincent et al., 2006). DotF and DotG are inner membrane proteins that interact with a subcomplex consisting of the putative outer membrane pore DotH and two associated lipoproteins DotC and DotD. Finally, a large number of the Dot/Icm proteins appear to be inner membrane proteins, including the type IV coupling protein DotL, and likely constitute the T4SS receptor for cytoplasmic substrates (see Fig. 3.1).

Outer membrane

DotH

Do

tC

tD

Do

tK

Do

Periplasm

DotF

DotA DotE DotI DotJ DotP DotV IcmT IcmV DotG

IcmW

DotL

M

DotU IcmF

Inner membrane

N IcmO IcmQ

Cytoplasm

DotB

IcmR ADP?

ATP? IcmS IcmW

FIGURE 3.1.

Substrates


53

Other components of the Dot/Icm system include the proteins DotU and IcmF, which have orthologs in a wide range of Gram-negative bacteria (Sexton et al., 2004a; VanRheenen et al., 2004). DotU and IcmF are thought to stabilize the T4SS to maintain an active form of the apparatus or protect it from degradation, as dotU and icmF mutants displayed decreased stability of the DotH/DotG/DotF subcomplex (Sexton et al., 2004a; VanRheenen et al., 2004). Surprisingly, over-expression of DotH was sufficient to suppress the destabilization of the Dot/Icm apparatus in the absence of DotU and IcmF, restoring normal levels of DotG and DotF, and eliminating the intracellular growth defect in the dotU/icmF mutant. This suggests that DotH is a key component of this subcomplex. Interestingly, DotU was shown to be degraded when L. pneumophila enters stationary phase (VanRheenen et al., 2004), a period where the organism shows increased virulence (Molofsky and Swanson, 2004). Thus, IcmF and DotU may function together to stabilize the T4SS in exponentially growing bacteria ensuring that the translocon becomes active at the correct point in the cell cycle (Sexton et al., 2004b; VanRheenen et al., 2004). Interestingly two Dot/Icm components, DotO and DotH, were observed on the surface of L. pneumophila after interaction with macrophages (Watarai et al., 2001). Since DotO and DotH surface expression correlated with the formation of a fibrous structure on the surface of L. pneumophila, it was proposed that this structure may play a critical role in the function of the Dot/Icm complex (Watarai et al., 2001).

2.2. Adaptor/Chaperone Complexes L. pneumophila icmS and icmW mutants had a unique phenotype compared to other dot/icm mutant strains since the LCV associated with vesicles, but then quickly fused with the endocytic pathway (Coers et al., 2000; Zuckman et al., 1999). IcmS and IcmW were both shown to bind to a number of Dot/Icm-secreted proteins, and to interact with each other, suggesting that they function as an adaptor complex involved in substrate recognition by the Dot/Icm complex (Bardill et al., 2005; Coers et al., 2000; Ninio et al., 2005). By analogy to the T3SS chaperones (Hueck, 1998), type IV adaptor complexes might bind secreted substrates to prevent their aggregation or degradation preceding export. Vincent and Vogel (2006) recently reported that the product of the Legionella virulence gene A (lvgA) was also involved in adaptor functions, and proposed that IcmS exists in two separate complexes (IcmS/IcmW and IcmS/LvgA), presumably selecting for distinct classes of effectors (Vincent and Vogel, 2006). This was consistent with a previous report (Bardill et al. 2005) where SdeA was shown to be secreted by a Dot/Icm-dependent process involving both DotA and IcmS, whereas another Dot/Icm-secreted factor RalF was translocated by a DotA-dependent, but IcmSindependent, mechanism (Bardill et al., 2005). Interestingly, lvgA mutants were only impaired for growth in A/J mouse macrophages and not in other macrophages or a protozoan host (Vincent and Vogel, 2006). Finally, IcmR was also shown to be a chaperone for IcmQ (Duménil and Isberg, 2001). Phagosomes harboring icmR mutants display another unique characteristic in that they do not

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recruit vesicles but are able to evade endocytic maturation (Coers et al., 2000). The different phenotypes observed between icmS or icmW and icmR mutants suggest that evasion of the endocytic pathway and formation of the LCV likely involve multiple Dot/Icm-secreted proteins whose functions are required at different stages of the infection process (Coers et al., 2000).

2.3. Regulation of Effector Export Before releasing effectors through the Dot/Icm secretion apparatus, L. pneumophila must create a pore in the host plasma membrane. Based on its ability to form pores in membranes, the IcmQ protein may be responsible for this activity (Duménil et al., 2004). Consistent with this notion, IcmQ appeared to be localized on the bacterial surface shortly after contact with host cells (Duménil et al., 2004). IcmQ-mediated pore formation appears to be inhibited via an interaction with the chaperone-like molecule, IcmR, thus providing a molecular mechanism of regulation (Duménil et al., 2004). A similar contact-dependent pore-forming activity has been observed in Shigella flexneri as a mechanism to control the release of effectors from its T3SS (Hueck, 1998). A second mechanism of regulation may be provided by the activity of DotL, the Dot/Icm type IV coupling protein (Buscher et al., 2005). The dotL gene has been found to be essential for viability in the Lp02 strain of L. pneumophila, but not JR32 (Buscher et al., 2005). ⌬dotL lethality can be suppressed by inactivation of other dot/icm genes, suggesting that the absence of DotL may result in the formation of toxic membrane complex(es). Consistent with this hypothesis, the JR32 strain of L. pneumophila tolerates a dotL mutation, but displays defects in ion homeostasis resulting in hypersensitivity to salt. Therefore, DotL appears to prevent the unregulated flow of solute (and presumably effectors) through the complex, consistent with a “gatekeeping” mechanism (Buscher et al., 2005). Gatekeeper functions have also been reported for the IpaB and IpaD effectors of S. flexneri, where ipaB and ipaD mutants resulted in unregulated secretion of substrates from the T3SS (Hueck, 1998). DotL, in conjunction with the type IV adaptors, likely provides the major control of secretion by the L. pneumophila T4SS.

3. Genetic Regulation of the Dot/Icm System Temporal regulation of both the Dot/Icm apparatus and its substrates has been reported. Transcriptional control of the Dot/Icm T4SS has been proposed to be mediated in part by the ppGpp synthase RelA (icmP), the stationary phase sigma factor RpoS (icmP and icmR), LetA/S (icmP, icmR, icmT, dotA), and CpxA/CpxR two-component systems (icmR, icmV, and icmW) (Gal-Mor et al., 2002; Gal-Mor and Segal, 2003a, b; Shi et al., 2006; Zusman et al., 2002). However, none of these regulatory factors are required for intracellular growth in macrophages. Moreover, expression of most components of the Dot/Icm system appears to be constitutive when assayed by western analysis (Bardill et al., 2005; Vincent and Vogel, 2006).


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For example, DotL, IcmS, and IcmW are clearly constitutively expressed in all phases of growth (Bardill et al., 2005; Vincent and Vogel, 2006). In contrast, a number of Dot/Icm substrates including SdeC, SidC, LidA, and RalF were shown to be highly expressed during the transmissive phase of growth (Bardill et al., 2005; Luo and Isberg, 2004; Nagai et al., 2002). Furthermore, using microarray and real-time quantitative PCR technologies, Brüggemann et al. (2006) demonstrated that dotA was expressed at all times during the biphasic life cycle of L. pneumophila in Acanthamoeba castellanii, whereas effector molecules (ralF, sdbB, sidC, sdcA, sidG, sdeA, sdeB, sdeC, sdhB, vipD, vipE, lidA, and sidE) were up-regulated during the transmissive phase of growth. However, it should be noted that other putative effector molecules (sdeD, sdbA, sdbC, lepA, and lepB) showed no expression differences during the intracellular life cycle.

4. Possible Effector Proteins and Associated Functions The L. pneumophila Dot/Icm T4SS was originally shown to be competent for secretion by its ability to mediate conjugal transfer of the plasmid RSF1010 (Segal and Shuman, 1998; Vogel et al., 1998). Interestingly, the presence of this plasmid substantially inhibited intracellular replication of L. pneumophila (Segal and Shuman, 1998) and thus might compete with native L. pneumophila substrates for interaction with the Dot/Icm system. Using a variety of molecular methods, over 50 proteins have been identified as putative substrates of the L. pneumophila Dot/Icm system. The best characterized are RalF, SidA-H, LidA, LepA-B, YlfA, and the Vip proteins.

4.1. RalF RalF, the recruitment of ARF to Legionella-phagosome factor, was the first identified native Dot/Icm substrate (Nagai et al., 2002). RalF contains a Sec7 domain, which functions as a guanine nucleotide exchange factor (GEF) for the Arf family of GTPases (Amor et al., 2005; Nagai et al., 2002). Dot/Icm-translocated RalF recruits and activates the host GTPase ADP-ribosylation factor (Arf)-1 to the LCV (Nagai et al., 2002). Since Arf1 is a well-characterized regulator of vesicular traffic, it was suggested that RalF might aid in the formation of the replicative phagosome. This was supported by evidence indicating that interference of Arf1 activity abrogated LCV biogenesis (Kagan and Roy, 2002; Derré and Isberg, 2004; Kagan et al., 2004). Moreover, over-expression of RalF in a yeast model system caused drastic growth defects, whereas a yeast strain expressing a RalF mutant (E103A) lacking Arf-GEF activity was able to grow at a rate similar to that of wild-type yeast cells (Campodonico et al., 2005). As a result, it was surprising when it was determined that ralF mutants were fully capable of evading the endocytic pathway, forming a wild-type replicative phagosome, and replicating to normal levels in both protozoa and macrophages (Nagai et al., 2002). This phenomenon, where Dot/Icm substrates are not essential for intracellular survival

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and replication, is not restricted to RalF but appears to be applicable to most, if not all, substrates. This is likely due to the high level of redundancy achieved by L. pneumophila via the export of many substrates.

4.2. The Sid Family The Sid “substrate of Icm/Dot” family of proteins were identified via a combination of a two-hybrid screen and a bacterial protein translocation assay (Luo and Isberg, 2004). In the original report one Sid protein, SidC, was localized to the cytoplasmic face of the LCV within macrophages (Luo and Isberg, 2004). Weber et al. (2006) recently reported that SidC and its paralog SdcA were able to specifically bind to phosphatidylinositol(4) phosphate [PI(4)P] found on the LCV, thus providing an explanation for why the proteins may be retained on the LCV. SidC and SdcA possess coiled-coil motifs that may act similarly to synaptosomalassociated protein receptors (SNAREs) to mediate protein–protein interactions involved during uptake and LCV biogenesis (Weber et al., 2006). Experimental confirmation that the Sid proteins and their paralogs are translocated into hosts remains to be done. One family of Sid proteins, the SidE family (SdeA, SdeB, SdeC), was independently identified via their interaction with the type IV adaptor protein IcmS (Bardill et al., 2005). SdeA was shown to be secreted into host cells via two independent assays, adenylate cyclase fusions and immunofluorescence colocalization, and export was dependent on a functional Dot/Icm complex and the adaptor protein IcmS (Bardill et al., 2005). Though the SidE family was shown to be translocated into host cells in a Dot/Icm-dependent manner, a quadruple mutant lacking sidE and its paralogs showed wild-type growth kinetics in macrophages and only a modest intracellular growth defect in A. castellanii (Bardill et al., 2005).

4.3. LidA and SidM The lidA gene was identified by Conover et al. (2003) as an insertional mutant that exhibited decreased viability in the presence of a functional Dot/Icm complex, i.e., “lowered viability in the presence of dot.” LidA may interact with DotL and modify its gatekeeper functions. After contact with host cells, LidA is translocated across into the host cell and localizes on the cytoplasmic face of the LCV similar to SidC. Due to the slower kinetics between RalF and LidA, it was suggested that once secretion of LidA was engaged, other substrates could also to be secreted (Conover et al., 2003). Curiously, LidA was maintained on the LCV throughout the infection process and possessed a coiled-coil domain, suggesting that it might interact with host proteins involved in LCV biogenesis (Derré and Isberg, 2005; Kagan and Roy, 2002). Over-expression of LidA resulted in the redistribution of the endoplasmic reticulum–Golgi intermediate compartment (ERGIC) and the disappearance of the Golgi compartment (Derré and Isberg, 2005). However, the disruptions seen with over-expression are likely not to be


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physiologically relevant and may be viewed as an over-exaggeration of LidA function since the ERGIC and the Golgi are normally intact during L. pneumophila replication. More recently, co-localization studies of Rab1 in infections using L. pneumophila sidM and lidA mutants suggested that LidA cooperates in the SidM-mediated recruitment of Rab1 (Machner and Isberg, 2006). Machner and Isberg (2006) demonstrated that the two translocated substrates, SidM and LidA, synergistically recruit the small GTPase Rab1 to the LCV in order to manipulate ER-to-Golgi traffic, thus recruiting early secretory vesicles necessary for LCV biogenesis. SidM possesses GEF activity that can stimulate Rab1 and exhibits higher activity than that observed for Arf1-stimulating GEF RalF (Machner and Isberg, 2006). Since Rab1 was shown to be crucial for LCV biogenesis (Derré and Isberg, 2004), the lack of an intracellular growth defect in macrophages infected with sidM mutants was surprising (Machner and Isberg, 2006). However, formation of mature replicative vacuoles was reduced with lidA mutants (Conover et al., 2003), perhaps due to LidA’s interaction with other small GTPases including Rab6 and Rab8 (Machner and Isberg, 2006).

4.4. LepA and LepB LepA and LepB were identified by sequence analysis the L. pneumophila genome for likely T4SS substrates and their export was confirmed to be Dot/Icm dependent (Chen et al., 2004). Typical of other Dot/Icm-substrates, inactivation of lepA, lepB, or both genes did not significantly diminish the intracellular replication of L. pneumophila within macrophages, although a lower survival rate was observed in a protozoan host (Chen et al., 2004). This phenotype appeared to be due to the non-lytic release of vesicles containing L. pneumophila from the amoeba. The release of L. pneumophila in vesicles has been described elsewhere (Berk et al., 1998; Rowbotham, 1986); however, factors involved in the process had not been described. Although the exact mechanism is unknown, host-specific effectors like LepA and LepB might play a valuable role in the environmental dissemination of L. pneumophila during the replicative cycle within its natural protozoan host.

4.5. Ylf and the Vips Using a Saccharomyces cerevisiae model, Campodonico et al. (2005) demonstrated that over-production of L. pneumophila yeast lethal factor (YlfA) resulted in vesicular trafficking defects, an effect attributed to its N-terminal coiled-coil regions. By sequence analysis, a paralog to YflA, YflB, was identified. Both YlfA and YlfB were shown to be secreted into host cells by a Dot/Icm-dependent process, although ylfA ylfB double mutants showed no intracellular growth defects in A. castellanii or murine macrophages. Unlike other Dot/Icm-secreted proteins (RalF, LidA, SdeC, and SidC) that co-localize on the LCV within the first 30 minutes of infection (Bardill et al., 2005, Conover et al., 2003; Luo and Isberg, 2004; Nagai et al., 2002), YlfA localizes to the LCV late in the infection process (Campodonico et al., 2005). It was suggested that YlfA may perform a

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more distant role in the cytoplasm after translocation rather than the direct modulation of the LCV itself (Campodonico et al., 2005). Also using a yeast model, Shohdy et al. (2005) identified L. pneumophila proteins that produced defects in the vacuole protein sorting (VPS) pathway in S. cerevisiae. Three vacuole protein sorting inhibitor proteins (Vips) that were able to alter vesicular trafficking were identified (Shohdy et al., 2005). The Vips shared no common structure and the vesicular trafficking defects differed, but they were all translocated via the Dot/Icm system (Shohdy et al., 2005). VipF had an acetyltransferase domain of unknown function and VipA contained a coiledcoil domain that could be involved in SNARE-mediated interactions (Shohdy et al., 2005). VipD was investigated in more detail since it had previously been identified as a factor that affected growth in phagocytic cells (VanRheenen et al., 2006). VipD contains a phospholipase domain homologous to ExoU, a T3SS effector in Pseudomonas aeruginosa that exhibits lipolytic activity (Rabin et al., 2006). Though VipD could be translocated into macrophages, no phospholipase activity could be detected (VanRheenen et al., 2006). Moreover, Shohdy et al. (2005) had previously shown that a fragment of VipD lacking the phospholipase domain interfered with the secretory pathway to a greater extent than the fulllength protein (Shohdy et al., 2005), that the C-terminal fragment of ExoU was sufficient to target to the host plasma membrane (Rabin et al., 2006), and therefore VipD may affect vesicular trafficking via its C-terminal coiled-coil domain instead of via its enzymatic domain. In summary, L. pneumophila exports a large number of substrates via the Dot/Icm type IV secretion system. These multiple effectors may act in concert to provide a vast repertoire of activities that perturbs or modifies host cell functions, thus allowing the LCV to avoid the endocytic pathway and mature into a replicate phagosome. Although the Dot/Icm system plays a key role in the modulation of host cell functions, mutants lacking single effector proteins do not have strong intracellular growth defects (Luo and Isberg, 2004; Ninio et al., 2005; Campodinico et al., 2005). This is likely due to functional redundancy and has necessitated the development of alterative approaches to determine their specific molecular function.

5. Temporal Modulation of the LCV Surface The identification of host pathways involved in LCV biogenesis may reveal which Dot/Icm substrates are crucial for intracellular growth. Based on this assumption, Dorer et al. (2006) used RNA interference in Drosophila Kc167 cells to inhibit multiple host trafficking pathways that were important for intracellular growth of L. pneumophila. This approach identified Cdc48/p97, a component of endoplasmic reticulum-associated degradation (ERAD), as playing an important role in the intracellular growth of L. pneumophila. Cdc48/p97 is an AAA-ATPase that forms a chaperone complex required for ERAD functions, where misfolded proteins from the ER are ubiquitinated, removed by


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Cdc48/p97, and delivered to the proteasome for degradation (Bar-Nun, 2005). Cdc48/p97 and poly-ubiquitinated proteins were found to localize to the LCV in a Dot/Icm-dependent process in both macrophages and Drosophila cells (Dorer et al., 2006). Specifically, the Cdc48/p97 chaperone complex appeared to be required for the timely removal of the Dot/Icm substrates LidA and SidC from the LCV (Dorer et al., 2006). Consistent with its involvement, inhibition of proteasome function led to decreased intracellular replication of L. pneumophila (Dorer et al., 2006). Thus, L. pneumophila seems to exploit the ubiquitination system to temporally modulate the surface composition of the LCV and promote a vacuole suitable for replication.

6. Conclusion The Dot/Icm secretion system plays a crucial role in the generation of a replication-permissive niche for L. pneumophila in a wide range of protozoan and mammalian hosts. Although a limited amount of work has been done to determine how this T4SS assembles and functions, much labor remains. Recent estimates indicate that the Dot/Icm system may export between 50 and 150 proteins making it one of the most robust bacterial secretion systems ever identified. Although the apparent functional redundancy of these substrates makes elucidation of their molecular function more challenging, it also provides fertile ground for future discoveries that will likely provide key information about host cell processes and pathogenesis.

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Weber, S. S., C. Ragaz, K. Reus, Y. Nyfeler, and H. Hilbi. 2006. Legionella pneumophila exploits PI(4)P to anchor secreted effector proteins to the replicative vacuole. PLoS Pathog 2:e46. Wiater, L. A., K. Dunn, F. R. Maxfield, and H. A. Shuman. 1998. Early events in phagosome establishment are required for intracellular survival of Legionella pneumophila. Infect Immun 66:4450–60. Zuckman, D. M., J. B. Hung, and C. R. Roy. 1999. Pore-forming activity is not sufficient for Legionella pneumophila phagosome trafficking and intracellular growth. Mol Microbiol 32:990–1001. Zusman, T., O. Gal-Mor, and G. Segal. 2002. Characterization of a Legionella pneumophila relA insertion mutant and toles of RelA and RpoS in virulence gene expression. J Bacteriol 184:67–75.

4 Life Cycle, Growth Cycles and Developmental Cycle of Legionella pneumophila Rafael A. Garduño

1. Introduction The Gram-negative bacterium L. pneumophila is an inhabitant of freshwater. Although free legionellae may be readily detected in freshwater, L. pneumophila is not a free-living organism but an intracellular parasite of amoebae (Fields 1996) that recently became an opportunistic human pathogen. Only accidentally, when water aerosol–containing L. pneumophila is inhaled or contaminated water is aspirated, does L. pneumophila enter the human lung to infect alveolar macrophages. In susceptible individuals (mainly immuno-compromised patients or the elderly), this initial infection may lead to an atypical pneumonia known as Legionnaires’ disease (Winn 1988, Weeratna et al. 1993). Legionnaires’ disease is always transmitted from the environment to humans, and thus constitutes an environmental disease that cannot be transmitted from person to person. A careful evaluation of the ecology of L. pneumophila would unequivocally lead to the conclusion that the accidental infection of humans is not advantageous to L. pneumophila, but rather a “lateral shuffle” (Spriggs 1987) leading to a dead end. Infection of the human host is a diversion from the natural life cycle of L. pneumophila in amoebae. In other words, it would be very difficult to argue that humans have played a defining role in the evolution of L. pneumophila. Despite years of accumulated clinical experience and intense epidemiological research, a satisfactory explanation for the lack of person-to-person transmission of Legionnaires’ disease does not exist. However, some recent studies that will be discussed below are beginning to provide explanations. Central to these studies has been the understanding of the life cycle and the developmental cycle of L. pneumophila. Given the recent advances in our understanding of L. pneumophila’s developmental cycle, I believe that the systematic study of the ecology and natural history of L. pneumophila will shed new light on the pathogenesis of L. pneumophila and the mechanism(s) by which Legionnaires’ disease is transmitted. The purpose of this chapter is dual. It intends to integrate and summarize what we know about the natural history of L. pneumophila. An ever-increasing body of literature continues to describe both the dramatic changes that L. pneumophila 65

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experiences as it grows in different environments, as well as its complex interactions with host cells. Regrettably, the terminology used in the literature to describe these changes and complex interactions is not consistent, and clarification of some concepts has become necessary. Therefore, this chapter also intends to clarify the concepts and definitions associated with the life cycle, the many growth cycles, and the developmental cycle of L. pneumophila.

2. The Life Cycle of L. pneumophila as Currently Understood Life cycles tell us a great deal about the natural history of a living organism. They are a synthesis of years of evolution, adaptation, specialization, and survival in nature. From an infectious disease standpoint, life cycles may provide clues to understand the mechanisms of pathogenesis of a particular parasite. The origin of pathogens and the evolution of virulence lie within the dominion of microbial ecology, and the life cycle of L. pneumophila surely is no exception. Amoebae are the primary natural hosts of L. pneumophila and constitute the central axis around which the life cycle of L. pneumophila revolves (Fig. 4.1). Rowbotham (1980) first recognized L. pneumophila to be an intracellular parasite of amoebae, shortly after the isolation and identification of the “Legionnaires’ disease bacterium” by McDade et al. (1977). Currently, ~40 species of Legionella have been identified (Benson and Fields 1998), all of which are able to parasitize amoebae. In addition, a large number of Legionella-like amoebal pathogens (Adeleke et al. 1996) that cannot be cultured in vitro demonstrate that Legionella has developed unique obligate intracellular relationships with specific amoebae. Finally, Legionella-related endosymbionts (known as X-bacteria) have reached a very specific mutual dependence with Amoeba proteus (Ahn et al. 1994, Jeon 2004). Collectively, these observations suggest that the genus Legionella is ancient, and very well adapted to amoebal hosts. Most likely, members of the genus Legionella (L. pneumophila included) first “learned” how to become intracellular parasites through frequent encounters with amoebae (Molmeret et al. 2005), and have since evolved to establish a wide spectrum of relationships with these hosts, from facultative intracellular and pathogenic to endosymbiotic. The amoebae–L. pneumophila relationship seems to occupy a place on the pathogenic side of the spectrum, as L. pneumophila often kills its host cells. The life cycle of L. pneumophila, first described by Rowbotham (1986) at the light microscope level, starts with the invasion of an amoebal host, and proceeds through a series of intracellular events that lead to the active replication of L. pneumophila and then the release of the bacterial progeny that infect new hosts (Fig. 4.1). As expected, the cycle begins and ends at the same point (i.e., infection of a new host). The following is a brief description of the specific events that constitute the life cycle of L. pneumophila and its progression through the amoebal host.

4. Life Cycle, Growth Cycles and Developmental Cycle Vesicle

Human

Mitochondrion Ribosome Vesicle Attachment and entry Establishment and differentiation

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Cycle reinitiation

1 7

Differentiation and replication

Passage through ciliates

2 3

8 6 Amoeba

5

Exit

Ciliate

4 Maturation

ER

MIF

Nucleus

RF Differentiation

Intermediate

FIGURE 4.1. The life cycle of L. pneumophila. The cycle begins with the attachment to and the invasion of an amoebal host. Post-internalization events include inhibition of phagosome–lysosome fusion and alteration of organelle traffic. L. pneumophila replicates in a ribosome-decorated vacuole that associates with the endoplasmic reticulum (ER) and acquires ER markers. After replication is finished the progeny exits the wasted amoeba to reinitiate the cycle. The life cycle of L. pneumophila is associated with bacterial differentiation. The two main morphological forms are the mature intracellular form, or MIF, and the replicative form, or RF, which differentiate into each other via intermediates. MIFs play a central role in the infection of amoebae and are the potential agents that spread Legionnaires’ disease to humans, either free or packaged into vesicles. MIF-ladden vesicles may be directly released by amoebae or indirectly by ciliates via an additional packaging step.

2.1. Attachment and Invasion Attachment of L. pneumophila to and the subsequent invasion of amoebae seem to involve several molecular players. A 170-kD galactose/N-acetylgalactosamine–inhibitable amoebal lectin (Gal/GalNAc lectin) seems to serve as a receptor for L. pneumophila, and be necessary for the invasion of Hartmanella vermiformis (Venkataraman et al. 1997). This receptor is de-phosphorylated upon the interaction of L. pneumophila with H. vermiformis, a process that requires contact with viable bacteria, suggesting that L. pneumophila plays an active role in the invasion process. Because invasion often depends on effective attachment, it is possible for the Gal/GalNAc lectin to serve as a primary binding site required to bring legionellae close to a secondary receptor that mediates invasion. However, the bacterial ligand for the Gal/GalNAc lectin has not been as yet identified. Although one may assume amoebae would actively seek bacteria to phagocytose them as a source of food, it has been determined that the association of

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legionellae to H. vermiformis is very poor, and that virulent L. pneumophila may actually mediate its own internalization (Fields et al. 1993). In support of the multifactorial nature of the attachment and invasion processes, several legionellae factors have been shown to be involved: RtxA (Cirillo et al. 2002), a type IV pilus known as CAP (for competence- and adherence-associated pili) (Stone and Abu Kwaik 1998), and a flagella-related factor (Bosshardt et al. 1997). The receptor for these legionellae ligands, and/or the mechanism by which they act, has not yet been determined. Similarly, it is still not clear what molecular mechanism amoebae deploy to internalize L. pneumophila (or what mechanism does L. pneumophila activate to mediate its internalization by amoebae) since inhibition of actin polymerization by cytochalasin D or disruption of microtubules by colchicine do not inhibit uptake of L. pneumophila (Harb et al. 1998, King et al. 1991). However, cytoskeletal proteins like paxillin and vinculin homologs are selectively dephosphorylated upon interaction of H. vermiformis with L. pneumophila (Venkataraman et al. 1998), suggesting that these proteins may be involved in the internalization of L. pneumophila. Morphologically, L. pneumophila has been seen entering amoebae by coiling phagocytosis (Bozue and Johnson 1996, Venkataraman et al. 1998). However, other forms of internalization (perhaps resembling receptor-mediated endocytosis or macropinocytosis) do occur and seem to be even more common than coiling phagocytosis (Abu Kwaik 1996, Fields 1993, Venkataraman et al. 1998). As implied by Harb et al. (1998) one should not assume that all amoebae use the same mechanisms to internalize L. pneumophila.

2.2. Post-Internalization Events and Intracellular Growth The morphological events that follow internalization of L. pneumophila by amoebae are similar to those thoroughly characterized in mammalian cells. These include the association of the legionellae-containing vacuole with vesicles and the rough endoplasmic reticulum, deformation of the vacuolar membrane to closely follow the contour of replicating legionellae, etc. In addition, biochemical methods and/or immunolabeling have shown that amoebal vacuoles containing L. pneumophila do not fuse with lysosomes, and later acquire endoplasmic reticulum (ER) markers (Bozue and Johnson 1996, Abu Kwaik 1996). It is within this specialized vacuole surrounded by the ER that L. pneumophila replicates. The same morphological and biochemical intracellular events described in Hartmanella and Acanthamoebae also happen in the social amoeba Dictyostelium discoideum (Hägele et al. 2000, Solomon et al. 2000). This social amoeba has emerged as an excellent model to study the L. pneumophila–host interaction, mainly because it is a haploid organism with a known genome sequence, and for which several genetic tools have been developed. Other chapters in this book provide extensive details about the intracellular events associated with L. pneumophila replication, and the effector proteins that mediate these events. Therefore, discussion of these topics is not needed here, except to say that the morphological similarities observed

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between intracellular events in mammalian cells and amoebae are substantiated by the fact that many L. pneumophila genes are required for infection of both amoebae and mammalian cells (Cianciotto and Fields 1992, Cirillo et al. 2002, Gao et al. 1997, Liles et al. 1999, Miyake et al. 2005, Polesky et al. 2001, Pruckler et al. 1995, Segal and Shuman 1999). However, L. pneumophila seems to exclusively use subsets of genes to establish intracellular infections in either mammalian cells (Gao et al. 1998) or amoebae (Brieland et al. 1997b).

2.3. End of Replication and Exit Once the host cell has been wasted after L. pneumophila replication, the bacterial progeny is released to the environment where new host cells are to be found. The life cycle is then completed. The killing of the host cell and the release of the bacterial progeny seem to be two related processes that are driven by L. pneumophila’s ability to form pores in membranes (Gao and Abu Kwaik 2000). A class of mini Tn10 insertion mutants defective in their pore-forming ability were unable to both kill infected Acanthamoeba polyphaga and exit the infected amoebae. These mutants remained inside live amoebae for up to 4 days, but did not continue to replicate. The demise of the amoebal host does not occur via apoptosis (Gao and Abu Kwaik 2000). An alternative way of exiting infected Acanthamoeba has been reported by Berk et al. (1998). Live L. pneumophila can be released within vesicles, likely as a consequence of the amoebae trying to get rid of their food vacuoles before encystation. In this process, vesicles containing hundreds of legionellae are produced, in contrast to the free legionellae released by the lytic process. Because these vesicles are of respirable-size, it has been argued that they could act as complex infectious units in the transmission of Legionnaires’ disease. Furthermore, legionellae inside vesicles seem more resistant to some environmental challenges (Berk et al. 1998), suggesting that this mode of exit is associated with additional fitness advantages. Interestingly, numerous legionellae-laden vesicles are also produced by the ciliate Tetrahymena (McNealy et al. 2002), as discussed below.

2.4. What Role Do Ciliates Play in the Life Cycle of L. pneumophila? In contrast to the clear role that amoebae play as natural hosts for the intracellular replication of L. pneumophila, the permissiveness of ciliates to L. pneumophila replication is dubious. Although a few studies have experimentally shown that the freshwater ciliate Tetrahymena pyriformis supports the multiplication of L. pneumophila at 30–35°C (Barbaree 1986, Fields et al. 1984, 1986, Steele and McLennan 1996), at 25°C T. pyriformis is not permissive (Fields et al. 1984). In addition, L. pneumophila could not grow in the ciliate Cyclidium in axenic culture (Barbaree 1986). Except for Legionella longbeachae, several other Legionella spp. (L. pneumophila included) were either unable to grow in T. pyriformis or showed inconsistent growth in this ciliate (Steele and McLennan 1996). Finally,

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Tetrahymena vorax did not support the intracellular growth of L. pneumophila at 20–22°C (Smith-Somerville 1991). In my lab, we have been unable to grow different L. pneumophila strains in Tetrahymena sp. incubated at ⱕ30°C. Therefore, ciliates do not seem to constitute the preferred host within which L. pneumophila multiplies in nature. Still, ciliates may play an important role in the life cycle of L. pneumophila, either supporting the growth of L. pneumophila in thermally altered waters, as reservoirs of L. pneumophila, or as producers of legionellaeladen vesicles (McNealy et al. 2002). In a tripartite co-culture including A. polyphaga, Tetrahymena sp, and a low concentration of L. pneumophila (⬍1 legionellae/amoebae), numerous legionellae-laden vesicles were produced. In contrast, no vesicles were observed in experiments with amoebae and legionellae, or in experiments with ciliates and legionellae, when the low concentration of L. pneumophila was used (McNealy et al. 2002). However, mixing L. pneumophila and ciliates at a 100:1 ratio, or higher, yields numerous legionellae-laden vesicles (S. G. Berk personal communication). It seems that amoebae can amplify a thinned population of legionellae, so that the thicker bacterial population can be then ingested by Tetrahymena and packaged into legionellae-laden vesicles that could act as potential infectious particles for humans (Rowbotham 1980) with associated fitness advantages as discussed above.

3. The Growth Cycles of L. pneumophila Legionella pneumophila is able to grow on agar plates as well as in nutrient-rich liquid media. The artificial growth of L. pneumophila in the lab underlines the point that L. pneumophila may follow a number of growth cycles that are not part of its natural life cycle described above. Yet, the multiple growth cycles of L. pneumophila are part of its biology and constitute helpful tools to better understand this bacterium. For instance, it is of great advantage that L. pneumophila can be coerced to grow in the lab, as this allows the application of routine genetic, biochemical, and physiological methods to its study. However, we should keep in mind that growth in the lab is irrelevant to the life cycle and natural history of L. pneumophila. The same principle applies to the growth of L. pneumophila in mammalian cells (e.g., human macrophages). Legionella pneumophila grows extracellularly in lab co-cultures with green unicellular algae and/or cyanobacteria (Hume and Hann 1984, Pope et al. 1982, Tison et al. 1980) or other bacterial species (Wadosky and Yee 1983). L. pneumophila respires and/or incorporates carbon compounds present in photosynthetic exudates, as determined with 14C-tracing experiments (Tison 1987). Extracellular growth of legionellae in association with other microorganisms may thus happen in nature, particularly in biofilms. However, the significance of this extracellular growth to the life cycle of L. pneumophila remains to be established, particularly because recent studies suggest that the only manner in which L. pneumophila generates progeny in biofilm systems is through intracellular growth in amoebae (Kuiper et al. 2004, Murga et al. 2001). Following this line of thought, some


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investigators have proposed that in its natural habitat the intracellular environment of protozoa is essential for L. pneumophila replication (Abu Kwaik et al. 1998, Fields 1993). In Fig. 4.2 the extracellular growth cycle of L. pneumophila is portrayed. For the reasons discussed above, this growth cycle is not a primary representation of the growth of L. pneumophila in environmental biofilms, but a representation of the growth of L. pneumophila in nutrient-rich broth or agar. As described by Byrne and Swanson (1998), the extracellular growth cycle alternates between exponential phase legionellae that actively replicate, and post-exponential phase legionellae that eventually stop replicating until placed in fresh medium where growth is reinitiated. There are clear developmental changes associated with the transition between exponential phase and post-exponential phase legionellae (e.g., changes in the ability to infect host cells), which will be discussed in Section 4 of this chapter. When it comes to growing intracellularly, L. pneumophila displays many growth cycles. Numerous mammalian cells in culture support the growth of

Broth or agar

1 2 Germination Extracellular cycle MIF Intermediate Replicative form or exponential phase form Virulent form or postexponential phase form

FIGURE 4.2. The extracellular growth cycle of L. pneumophila. L. pneumophila enters the extracellular growth cycle via differentiation of MIFs into replicative forms in a nutrientrich medium. The presence of amino acids seems to be the trigger for germination. This step occurs through morphological intermediates. The replicative form is non-infectious to cells and cannot initiate intracellular growth cycles. As the nutrients are exhausted, cell division stops and the replicative form differentiates into a virulent form that stays in a stationary growth phase until passed into fresh nutrient-rich media. The virulent form can initiate intracellular growth cycles.

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L. pneumophila, some of which have been listed by Fields (1996). Technically, each of these cultured mammalian cells is associated with a unique L. pneumophila growth cycle that shows similarities and differences with both the L. pneumophila life cycle that primarily evolved in amoebae, and with growth cycles in other host cells. Many of the differences occur in the early stages of infection, e.g., attachment and invasion, whereas many of the similarities are found in the post-internalization and replication stages. The similarities and differences between growth in mammalian cells and amoebae have been highlighted in previous reviews (e.g., Fields 1996, Hoffman and Garduño 1999). Whole mammals (e.g., mice, guinea pigs) and embryonated chicken eggs also support the growth of L. pneumophila. These animals constitute very valuable models for understanding the human infection, but as previously stated in relation to the infection of humans, infection of these animals is irrelevant for the life cycle and the natural history of L. pneumophila. If L. pneumophila displays as many growth cycles as there are hosts, an intriguing point is whether L. pneumophila possesses a plethora of molecular factors that allow it to gain access to and replicate in these many hosts, or whether L. pneumophila cleverly exploits a very fundamental, ancient mechanism that is common to many eukaryotic cells. The current experimental evidence available, as presented above, could be interpreted one way or another, leaving the issue undecided. However, the many differences and variations that characterize the attachment to and entry of L. pneumophila into cells (Cirillo et al. 2000, Garduño et al. 1998a,b, Gibson et al. 1994, Harb et al. 1998, Payne and Horwitz 1987) seem to favor the notion that L. pneumophila has developed many different invasion factors. How did these factors evolve in the context of frequent interactions with amoebae (Molmeret et al. 2005) is not clear, but a subject of interest to understand the evolution of intracellular parasitism.

4. The Developmental Cycle of L. pneumophila The life cycle of L. pneumophila is accompanied by changes in the bacterium, some of which are very dramatic. First noticed by Rowbotham (1986) were the morphological changes in the progeny that emerges from wasted amoeba trophozoites. Besides being shorter than legionellae grown in vitro, the rods released by infected amoebae were also transiently motile. Through the years, it has become clear that growth of L. pneumophila in amoebae is also associated with changes in biochemistry, physiology, and virulence potential. L. pneumophila pre-grown in amoebae has the following characteristics: • • • • • • •

Enhanced resistance to chemicals (biocides) (Barker et al. 1992) Altered fatty acid profile, and possibly altered LPS (Barker et al. 1993) Altered protein profile (Barker et al. 1993, Cirillo et al. 1994) Bright red Giménez staining (Cirillo et al. 1994) Shorter size (Barker et al. 1995, Cirillo et al. 1994, Rowbotham 1986) Increased infectivity to mammalian cells and amoebae (Cirillo et al. 1994) Enhanced resistance to antibiotics (Barker et al. 1995)


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• Increased environmental fitness (Abu Kwaik et al. 1997) • Increased uptake by coiling phagocytosis (Cirillo et al. 1994, 1999) • Increased virulence to mice (Cirillo et al. 1999, Brieland et al. 1997a) Such profound and numerous changes are compatible with bacterial differentiation. At the time when some of the above phenotypes were characterized, it was also determined that they were lost upon subculture of L. pneumophila in lab media (and of course regained upon growth in amoebae), suggesting that L. pneumophila had gone through a reversible transition compatible with a cyclic change. It was in the context of the growth cycle in HeLa cells that the cyclical ultrastructural changes that L. pneumophila experiences during its intracellular growth were observed and studied (Garduño et al. 1998b, 2002a). These studies benefited from working with a highly pure population of HeLa cell-grown L. pneumophila, isolated from legionellae-laden vacuoles and HeLa cell debris by differential centrifugation in continuous density gradients (Garduño et al. 1998b). Actively replicating legionellae inside ribosome-decorated vacuoles were seen undergoing cell division, and displayed a typical Gram-negative envelope ultrastructure. In contrast, purified HeLa cell-grown L. pneumophila were never seen undergoing cell division, and presented a distinct thickening of the inner leaflet of the outer membrane and long invaginations of the cytoplasmic membrane (Garduño et al. 1988b, 2002b). HeLa cell-grown legionellae also showed several of the features listed above for amoebae-grown L. pneumophila including enhanced resistance to chemicals (detergents), an altered protein profile, a bright red Giménez staining, shorter size, increased infectivity to mammalian cells, enhanced resistance to antibiotics, and increased environmental fitness (Garduño et al. 1988b, 2002a, 2002b). Collectively, our HeLa cell results and those obtained in amoebae [mainly by Rowbotham (1986)] indicated a clear alternation between different forms of L. pneumophila that strongly suggested the existence of a developmental cycle. A detailed ultrastructural study (Faulkner and Garduño 2002) further established the cyclical morphological development that L. pneumophila follows when it differentiates along the HeLa cell growth cycle (modeled in Fig. 4.3). Recently, the existence of different L. pneumophila developmental forms was confirmed at the ultrastructural level in H. vermiformis (Greub and Raoult 2003), but even in past literature these forms are easily distinguished in several electron micrographs of infected amoebae, as discussed by Faulkner & Garduño (2002). Therefore, the life cycle of L. pneumophila, as some intracellular growth cycles in cultured cells, is associated with defined morphological, biochemical, and physiological changes that define a developmental cycle along which L. pneumophila differentiates.

4.1. Legionella pneumophila Development Along the Extracellular Growth Cycle The extracellular growth cycle of L. pneumophila is also associated with a differentiation process, initially described by Byrne and Swanson (1998). The post-exponential form (or stationary phase form) of L. pneumophila (Fig. 4.2)

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Mitochondrion Ribosome Vesicle or

HeLa MIF

ER

Nucleus

Intermediate Replicative form Virulent form or postexponential phase form

FIGURE 4.3. The growth cycle of L. pneumophila in HeLa cells. The cycle begins with the attachment to and the invasion of a HeLa cell, a process mediated (at least partially) by the L. pneumophila chaperonin. Cell infection is initiated by stationary phase forms, or by MIFs, because replicative forms do not proceed to establish an intracellular infection (patterned arrow with the “do not” sign). Post-internalization events are very similar to those observed in amoebae, and L. pneumophila also replicates in a ribosome-decorated vacuole that associates with the endoplasmic reticulum (ER) and acquires ER markers. After replication is finished, the progeny exits the wasted HeLa cell either as free MIFs or as large MIF-ladden vesicles. The intracellular growth cycle in HeLa cells is associated with bacterial differentiation, whereas the intracellular growth cycle in macrophages does not lead to the efficient production of MIFs.

is sodium sensitive, cytotoxic to mammalian cells, osmotically resistant, motile, infectious to cultured mouse macrophages, and capable of inhibiting phagosome–lysosome fusion. All these characteristics were absent in the exponential phase form (Byrne and Swanson 1998), which has been further confirmed to be non-infectious to cells in culture (Joshi et al. 2001). This clear distinction between the two forms of the extracellular growth cycle led to the proposed names by Byrne and Swanson (1998) of “replicative form” for the exponential phase form, and “virulent form” for the post-exponential phase form. The regulatory system that controls L. pneumophila differentiation in the extracellular growth cycle has been studied at the molecular level, mainly in the Swanson lab. As comprehensibly reviewed by Molofsky and Swanson (2004), entry into stationary phase is triggered by the activation of the stringent response regulator RelA and accumulation of the molecular alarmone ppGpp (Hammer and Swanson 1999). To coordinate the expression of virulence traits and differentiation, ppGpp and RelA act in concert with the following molecular players:


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• The two-component regulatory system LetA/S (responsible for signal transduction with help from LetE) (Bachman and Swanson 2004, Hammer et al. 2002) • The sigma factors RpoS, RpoN, and FliA (Bachman and Swanson 2001, Hales and Shuman 1999, Hammer et al. 2002, Heuner et al. 2002, Zussman et al. 2002) • CsrA (a repressor of mRNA translation) (Forsbach-Birk et al. 2004, Molofsky and Swanson 2003). However, the role of RelA may be redundant and/or not essential because L. pneumophila relA mutants still induce cytotoxicity and keep RpoS-dependent functions (Abu-Zant et al. 2006), also suggesting a lack of control of ppGpp levels and the stringent response over RpoS function (Abu-Zant et al. 2006, Zussman et al. 2002). A key question would be: Is the developmental program that gives rise to the post-exponential virulent form in vitro the same that L. pneumophila uses to differentiate in amoebae and HeLa cells? In a head-to-head experimental comparison, HeLa cell-grown legionellae and post-exponential forms from the same L. pneumophila strain grown in vitro had different phenotypes (Garduño et al. 2002b). Post-exponential forms were less infectious to mammalian cells in culture, less resistant to detergents or antibiotics, and had a higher respiration rate than HeLa cell-grown legionellae. Indirect comparison with published data also indicated that post-exponential virulent forms are different from amoebae-grown legionellae (e.g., Cirillo et al. 1994, and Byrne and Swanson 1998 vs. Barker et al. 1992, 1995). However, post-exponential phase and HeLa cell-grown forms share a number of traits, like thermal tolerance, resistance to osmotic shock and protease treatments, and production of cytoplasmic inclusions. Moreover, mutants with a defective LetA/S two component regulatory system, or with a deleted rpoS gene, which are defective in developing the post-exponential virulent phenotype, are also unable to develop the wild type characteristics of HeLa cell-grown legionellae (unpublished results). Finally, post-exponential virulent forms develop the unique morphological features of HeLa cell- or amoebaegrown legionellae upon interaction with the ciliate Tetrahymena, and in the absence of intracellular multiplication (unpublished results). Collectively, these results suggest that (i) L. pneumophila contains a developmental program, (ii) a known trigger of this developmental program (among others) is amino acid starvation, (iii) the developmental program of L. pneumophila involves a regulatory network of which LetA/S and RpoS are essential members, and (iv) the post-exponential virulent form developed in vitro constitutes a partially differentiated form that in the right conditions can further differentiate. By reasons not yet understood, full differentiation into the highly infectious and resilient developmental form reported by Barker et al. (1992, 1993, 1995), Cirillo et al. (1994, 1999), Abu Kwaik et al. (1997), and Garduño et al. (2002b) seems to only take place in the intracellular environment of L. pneumophila hosts. Therefore, in addition to amino acid starvation, other signals seem to be required for full differentiation of L. pneumophila. In spite of representing an immature L. pneumophila form that does not express a full virulence

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phenotype, post-exponential phase forms have served as a practical tool to study L. pneumophila differentiation at the molecular level, and to elucidate the complex regulatory pathway that controls the expression of virulence traits (Molofsky and Swanson 2004).

4.2. MIF: The Result of L. pneumophila Intracellular Differentiation Intracellularly, L. pneumophila possesses two main morphological forms, the replicative form (RF) and the mature intracellular form (MIF), which alternate within the developmental cycle of L. pneumophila via several morphological intermediates (Faulkner and Garduño 2002, Greub and Raoult 2003). The RF actively undergoes cell division, has the typical ultrastructure of Gram-negative bacteria (defined by two membranes and a periplasmic space between them), and does not persist in the extracellular environment. The RF seems equivalent to the replicative form, or exponential phase form characterized by Byrne and Swanson (1998), which is non-infectious (Joshi et al. 2001). In contrast, the MIF does not undergo cell division, has a complex laminated envelope defined by several membranes and layers, displays prominent intracellular inclusions, and is highly infectious to cells in culture and mice (Cirillo et al. 1994, 1999, Garduño et al. 2002b). The MIF is the intracellular equivalent of the postreplicative form, which exits infected cells and persists in the environment. If we accept the notion that in its natural habitat L. pneumophila only replicates in the intracellular environment (see Section 3 above), then we are compelled to accept the notion that the MIF is the L. pneumophila free form that predominates in the environment. That would explain why MIFs are environmentally resilient and highly infectious, loaded with inclusions of storage polymers, and exhibit a very low respiration rate. The MIF is the specialized form of L. pneumophila designed to survive in the environment for several months (James et al. 1999, Lee and West 1991, Schofield 1985, Skaliy and McEachern 1979), and assail potential hosts. Once in the intracellular environment, MIFs differentiate (or “germinate,” to use the same terminology introduced in Fig. 4.2) into RFs, likely in response to an elevated concentration of amino acids putatively transported from the host cell cytoplasm into the replicative vacuole (Sauer et al. 2005). After L. pneumophila replication, and in response to amino acid starvation and other signals, RFs differentiate into MIFs in the intracellular environment. MIFs then exit the wasted host cell and disperse in the environment until a new host is found. Clearly, the MIF-to-RF and RF-to-MIF transitions naturally occur in the intracellular environment, and MIFs naturally constitute the resting cyst-like forms that survive free in the water environment. Whether MIFs are capable to further differentiate (e.g., to produce the viable but non-culturable form of L. pneumophila) remains to be established. One remarkable observation is the fact that MIFs seem inconspicuous in cultured human macrophages (Garduño et al. 2002b). Even in a retrospective analysis of past literature, MIFs could not be clearly identified in micrographs of infected


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macrophages. It is very possible that upon inducing early apoptosis in macrophages (Gao and Abu Kwaik 1999, Hägele et al. 1998), L. pneumophila cuts short its time window available to achieve full differentiation. Alternatively, the signals that lead to full differentiation of L. pneumophila into MIFs may be present in amoebae or in non-lymphoid mammalian cell lines, but absent in macrophages. Assuming that the MIF is the transmissible form of L. pneumophila in nature, the fact that MIFs scantily form in human macrophages (Garduño et al. 2002b) may provide the first clue to explain the environmental quality of Legionnaires’ disease, and its lack of transmission from person to person.

4.3. The Central Role of MIFs in the Study of L. pneumophila Virulence The MIF seems to hold the key to understand the transmission and infectiousness of L. pneumophila. First, in contrast to the non-infectious RF, the MIF is infectious to amoebae, a number of cells cultured in vitro, and mice. The previously reported virulence-enhancing role of amoebae (Brieland et al. 1996, 1997a, 1997b, Cirillo et al. 1994, 1999) is most likely due to the developmental differentiation of L. pneumophila into MIFs inside amoebae. Thus, MIFs seem to play a central role in the infection of both amoebae and mammalian hosts. In the L. pneumophila life cycle, MIFs in water infect amoebae, become established in the intracellular environment, and differentiate into RFs. However, when released from wasted amoebae, L. pneumophila emerges again as MIFs either free (as a result of necrotic lysis of the host) or packaged into vesicles (as described by Berk et al. 1998). Most likely, MIFs released from amoebae are the ones accidentally diverted into the human host to initiate Legionnaires’ disease in susceptible individuals. Finally, free MIFs emerging from infected amoebae are the ones packaged by ciliates into respirable-size vesicles (McNealy et al. 2002) that also may accidentally initiate human infections. MIFs must possess all the factors required to survive in water and infect new hosts. This prediction is based on the facts that MIF’s metabolism is low (Garduño et al. 2002b), and that invasion of new hosts and the intracellular establishment of L. pneumophila takes but minutes after the initial contact (Lu and Clarke 2005, Roy et al. 1998). It is our belief that within this narrow frame there is not enough time for the metabolically challenged MIF to mount a de novo response to the host cell, start the developmental program of differentiation into the RF, initiate a rapid synthesis of virulence effectors, assemble a functional Dot/Icm secretion system, and deliver effectors to the host cell. Instead, it is appealing to view MIFs as preloaded units carrying all the goods required to assure a successful infection of new hosts. In this respect, Roy et al. (1998) demonstrated, by placing the dotA gene under the control of an inducible promoter, that DotA was required for invasion and intracellular establishment, but was dispensable during intracellular growth (the RF phase). When dotA expression was “turned off” toward the end of the intracellular growth cycle, the resulting progeny could not re-infect cells, elegantly suggesting that

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the bacteria emerging from a wasted host cell (i.e., MIFs) must already have a functional Dot/Icm system to assure infection of new hosts. The expression of other virulence-related proteins (e.g., DotO, DotH, and Hsp60) in the late stages of intracellular growth (corresponding to differentiation of RFs into MIFs) but not in the post-exponential phase of the extracellular growth cycle (Garduño et al. 2002b, Watarai et al. 2001) points to the unique array of virulence factors that equip MIFs to best perform their duty: the efficient assailment of new hosts.

5. Concluding Remarks It seems clear now that L. pneumophila has a defined developmental cycle embedded in its life cycle. Developmental cycles are central to the pathogenesis and ecology of obligate intracellular bacterial pathogens with an extracellular phase. Therefore, differentiation into a highly infectious and environmentally resilient form that survives extended periods of starvation in freshwater (just to have the opportunity to gain access into new hosts) constitutes a trait evolved by L. pneumophila that has become an integral part of its natural history. The term “life cycle” should only be used in reference to the growth cycle and differentiation of L. pneumophila in amoebae and other protozoa. Similarly, the term “MIF” should only be used to refer to the differentiated L. pneumophila form that emerges from protozoa, or from mammalian cells that support the full differentiation of L. pneumophila (e.g., HeLa cells). MIFs should be regarded as the transmissive forms of L. pneumophila. Post-exponential virulent forms grown in vitro, unlikely to exist in nature, should not be regarded as the transmissive form of L. pneumophila in nature. In vitro grown legionellae should be held in the highest regard as experimental models to study aspects of L. pneumophila development and its general biology. In many respects, the L. pneumophila life cycle resembles the life cycles of Chlamydia and Coxiella, which are obligate intracellular pathogens that spread via cyst-like forms. In particular, Coxiella burnetti is a bioterrorism agent (CDC list of bioterrorism agents: http://www.bt.cdc.gov/agent/agentlist.asp) that shows striking similarities with Lp: • It has a dimorphic developmental cycle (Coleman et al. 2004). • It is highly infectious by aerosol (Glazunova et al. 2005). • It exploits autophagy (as L. pneumophila does) to establish intracellular infections (Amer and Swanson 2005, Gutierrez et al. 2005). • It possesses similar virulence factors including the Dot/Icm system (Vogel 2004). The complete DNA sequence of its genome has confirmed Coxiella burnetti to be the closest relative of L. pneumophila (Samuel et al. 2003, Vogel 2004). The facts that L. pneumophila grows in vitro (either in broth or on agar plates) and only requires level-2 biohazard containment (in contrast to Coxiella which is an


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obligate intracellular pathogen that cannot grow in vitro, and requires a level-3 containment) are significant advantages for its use as an experimental surrogate for Coxiella. Thus, MIFs have the potential of becoming the preferred experimental model to study intracellular bacteria transmitted by cyst-like forms.

References Abu Kwaik Y (1996) The phagosome containing Legionella pneumophila within the protozoan Hartmannella vermiformis is surrounded by the rough endoplasmic reticulum. Appl Environ Microbiol 62:2022–2028. Abu Kwaik Y, Gao L-Y, Harb OS, and Stone BJ (1997) Transcriptional regulation of the macrophage-induced gene (gspA) of Legionella pneumophila and phenotypic characterization of a null mutant. Mol Microbiol 24:629–642. Abu Kwaik Y, Gao L-Y, Stone BJ, Venkataraman C, and Harb OS (1998) Invasion of protozoa by Legionella pneumophila and its role in bacterial ecology and pathogenesis. Appl Environ Microbiol 64:3127–3133. Abu-Zant A, Asare R, Graham JE, and Abu Kwaik Y (2006) Role of RpoS but not RelA of Legionella pneumophila in modulation of phagosome biogenesis and adaptation to the phagosomal microenvironment. Infect Immun 74:3021—3026. Adeleke A, Pruckler J, Benson R, Rowbotham T, Halablab M, and Fields B (1996) Legionella-like amoebal pathogens—Phylogenetic status and possible role in respiratory disease. Emerg Infect Dis 2:225–230. Ahn TI, Lim ST, Leeu HK, Lee JE, and Jeon KW (1994) A novel strong promoter of the groEx operon of symbiotic bacteria in Amoeba proteus. Gene 148:43–49. Amer AO, and Swanson MS (2005) Autophagy is an immediate macrophage response to Legionella pneumophila. Cell Microbiol 7:765–778. Bachman MA, and Swanson MS (2001) RpoS co-operates with other factors to induce Legionella pneumophila virulence in the stationary phase. Mol Microbiol 40:1201–1214. Bachman MA, and Swanson MS (2004) The LetE protein enhances expression of multiple LetA/LetS-dependent transmission traits by Legionella pneumophila. Infect Immun 72:3284–3293. Barbaree JM, Fields BS, Feeley JC, Gorman GW, and Martin WT (1986) Isolation of protozoa from water associated with a legionellosis outbreak and demonstration of intracellular multiplication of Legionella pneumophila. Appl Environ Microbiol 51:422–424. Barker J, Brown MRW, Collier PJ, Farrell I, and Gilbert P (1992) Relationship between Legionella pneumophila and Acanthamoeba polyphaga: physiological status and susceptibility to chemical inactivation. Appl Environ Microbiol 58:2420–2425. Barker J, Lambert PA, and Brown MR (1993) Influence of intra-amoebic and other growth conditions on the surface properties of Legionella pneumophila. Infect Immun 61:3503–3510. Barker J, Scaife H, and Brown MR (1995) Intraphagocytic growth induces an antibioticresistant phenotype of Legionella pneumophila. Antimicrob Agents Chemother 39:2684–2688. Benson RF, and Fields BS (1998) Classification of the genus Legionella. Semin Respir Infect 13:90–99. Berk SG, Ting RS, Turner GW, and Ashburn RJ (1998) Production of respirable vesicles containing live Legionella pneumophila cells by two Acanthamoeba spp. Appl Environ Microbiol 64:279–286.

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Bosshardt SC, Benson RF, and Fields BS (1997) Flagella are a positive predictor for virulence in Legionella. Microb Pathogen 23:107–112. Bozue, JA, and Johnson W (1996) Interaction of Legionella pneumophila with Acanthamoeba castellani: Uptake by coiling phagocytosis and inhibition of phagosomelysosome fusion. Infect Immun 64:668–673. Brieland J, McClain M, Heath L, Chrisp C, Huffnagle G, LeGendre M, Hurley M, Fantone J, and Engleberg C (1996) Coinoculation with Hartmanella vermiformis enhances replicative Legionella pneumophila lung infection in a murine model of Legionnaires’ disease. Infect Immun 64:2449–2456. Brieland JK, Fantone JC, Remick DG, LeGendre M, McClain M, and Engleberg NC (1997a) The role of Legionella pneumophila-infected Hartmanella vermiformis as an infectious particle in a murine model of Legionnaires’ disease. Infect Immun 65:5330–5333. Brieland J, McClain M, LeGendre M, and Engleberg C (1997b) Intrapulmonary Hartmannella vermiformis: a potential niche for Legionella pneumophila replication in a murine model of legionellosis. Infect Immun 65:4892–4896. Byrne B, and Swanson MS (1998) Expression of Legionella pneumophila virulence traits in response to growth conditions. Infect Immun 66:3029–3034. Cianciotto NP, and Fields BS (1992) Legionella pneumophila mip gene potentiates intracellular infection of protozoa and human macrophages. Proc Natl Acad Sci USA 89:5188–5191. Cirillo JD, Falkow S, and Tompkins LS (1994) Growth of Legionella pneumophila in Acanthamoeba castellani enhances invasion. Infect Immun 62:3254–3261. Cirillo JD, Cirillo SLG, Yan L, Bermudez LE, Falkow S, and Tompkins LS (1999) Intracellular growth in Acanthamoeba castellani affects monocyte entry mechanisms and enhances virulence of Legionella pneumophila. Infect Immun 67:4427–4434. Cirillo SLG, Lum J, and Cirillo JD (2000) Identification of novel loci involved in entry by Legionella pneumophila. Microbiology 146:1345–1359. Cirillo SLG, Yang L, Littman M, Samrakandi MM, and Cirillo JD (2002) Role of the Legionella pneumophila rtxA gene in amoebae. Microbiology 148:1667–1677. Coleman SA, Fischer ER, Howe D, Mead DJ, and Heinzen RA (2004) Temporal analysis of Coxiella burnetii morphological differentiation. J Bacteriol 186:7344–7352. Faulkner G, and Garduño RA (2002) Ultrastructural analysis of differentiation in Legionella pneumophila. J Bacteriol 184:7025–7041. Fields BS (1993) Legionella and protozoa: interaction of a pathogen and its natural host. In: Barbaree JM, Breiman RF, Dufour AP (eds.) Legionella: Current status and emerging perspectives. American Society of Microbiology, Washington, pp 129–136. Fields BS (1996) Molecular ecology of legionellae. Trends Microbiol 4:286–290. Fields BS, Shotts EB Jr, Feeley JC, Gorman GW, and Martin WT (1984) Proliferation of Legionella pneumophila as an intracellular parasite of the ciliated protozoan Tetrahymena pyriformis. Appl Environ Microbiol 47:467–471. Fields BS, Barbaree JM, Shotts EB Jr, Feeley JC, Morrill WE, Sanden GN, and Dykstra MJ (1986) Comparison of guinea pig and protozoan models for determining virulence of Legionella species. Infect Immun 53:553–559. Fields BS, Fields SRU, Loy JNC, White EH, Steffens WL, and Shotts EB (1993) Attachment and entry of Legionella pneumophila in Hartmanella vermiformis. J Infect Dis 167:1146–1150. Forsbach-Birk V, McNealy T, Shi C, Lynch D, and Marre R (2004) Reduced expression of the global regulator protein CsrA in Legionella pneumophila affects


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Harb OS, Venkataraman C, Haack BJ, Gao L-Y, and Abu Kwaik Y (1998) Heterogeneity in the attachment and uptake mechanisms of the Legionnaires’ disease bacterium, Legionella pneumophila, by protozoan hosts. Appl Environ Microbiol 64: 126–132. Heuner K, Dietrich C, Skriwan C, Steinert M, and Hacker J (2002) Influence of the alternative sigma-28 factor on virulence and flagellum expression of Legionella pneumophila. Infect Immun 70:1604–1608. Hoffman PS, and Garduño RA (1999) Pathogenesis of Legionella pneumophila infection. In: Paradise LJ, Friedman H, and Bendinelli M (eds) Opportunistic Intracellular Bacteria and Immunity. Plenum Press, New York, pp. 131–147. Hume RD, and Hann WD (1984) Growth relationships of Legionella pneumophila with green algae (Chlorophyta). In: Thornsberry C, Balows A, Feeley JC, and Jakubowski W (eds) Legionella, Proceedings of the 2nd International Conference. American Society of Microbiology, Washington, pp. 323–324. James BW, Mauchline WS, Dennis PJ, Keevil CW, and Wait R (1999) Poly-3-hydroxybutyrate in Legionella pneumophila, an energy source for survival in low-nutrient environments. Appl Environ Microbiol 65:822–827. Jeon KW (2004) Genetic and physiological interactions in the amoeba-bacteria symbiosis. J Eukaryot Microbiol 51:502–508. Joshi AD, Sturgill-Koszycki S, and Swanson MS (2001) Evidence that Dot-dependent and -independent factors isolate the Legionella pneumophila phagosome from the endocytic network in mouse macrophages. Cell Microbiol 3:99–114. King CH, Fields BS, Shotts EB Jr, and White EH (1991) Effects of cytochalasin D and methylamine on intracellular growth of Legionella pneumophila in amoebae and human monocyte-like cells. Infect Immun 59:758–763. Kuiper MW, Wullings BA, Akkermans ADL, Beumer RR, and van der Kooij D (2004) Intracellular proliferation of Legionella pneumophila in Hartmanella vermiformis in aquatic biofilms grown on plasticized polyvinyl chloride. Appl Environ Microbiol 70:6826–6833. Lee JV, and West AA (1991) Survival and growth of Legionella species in the environment. J Appl Bacteriol (Symp Suppl) 70:121S–129S. Liles MR, Edelstein PH, and Cianciotto NP (1999) The prepilin peptidase is required for protein secretion by and the virulence of the intracellular pathogen Legionella pneumophila. Mol Microbiol 31:959–970. Lu H, and Clarke M (2005) Dynamic properties of Legionella-containing phagosomes in Dictyostelium amoebae. Cell Microbiol 7:995–1007. McDade JE, Shepard CC, Fraser DW, Tsai TR, Redus MA, Dowdle WR, et al. (1977) Isolation of a bacterium and demonstration of its role in other respiratory disease. N Engl J Med 297:1197–1203. McNealy T, Newsome AL, Johnson RA, and Berk SG (2002) Impact of amoebae, bacteria and Tetrahymena on Legionella pneumophila multiplication and distribution in an aquatic environment. In: Marre R, AbuKwaik Y, Bartlett C, Cianciotto NP, Fields BS, Frosch M, Hacker J, and Lück PC (eds.) Legionella. ASM Press, Washington, pp. 170–175. Miyake M, Watanabe T, Koike H, Molmeret M, Imai Y, and Abu Kwaik Y (2005) Characterization of Legionella pneumophila pmiA, a gene essential for infectivity of protozoa and macrophages. Infect Immun. 73:6272–6282. Molmeret M, Horn M, Wagner M, Santic M, and Abu Kwaik Y (2005) Amoebae as training grounds for intracellular bacterial pathogens. Appl Environ Microbiol 71:20–28.


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5 Legionella in the Environment Barry S. Fields

1. Introduction Legionellae survive in environments with their primary hosts, free-living protozoa. These bacteria probably survive in many of the same environments inhabited protozoa although the full range of these environments is unknown. Legionellae bacteria are well suited to aquatic environments and freshwater is the primary reservoir for legionellae worldwide (Fliermans et al.1981). Although legionellae can be found in waters ranging from cold to very hot, their multiplication is limited to temperatures between 25 and 42°C with an optimal growth at 35°C. Therefore the bacteria colonize and persist in moist environments within this temperature range whereas their presence in other environments is probably transitory. Some outbreaks of legionellosis have been associated with construction, and it was originally believed that the bacteria could survive and be transmitted to humans via soil. However, legionellae do not survive in dry environments, and these outbreaks are more likely the result of massive descalement of plumbing systems due to changes in water pressure during construction (Katz and Hammel 1987; Mermel et al. 1995).

2. The Distribution of Legionella as a Function of Various Detection Methods Descriptions of the prevalence and distribution of legionella depends upon the detection method used. Results from culture-independent methods suggest a greater distribution and concentration of legionellae in the environment. These methods offer the potential of greatly increased sensitivity. However, culture remains the method of choice for detecting legionellae, primarily because cultureindependent methods cannot provide information regarding the viability of the bacteria. These non-culture methods include detection of the organisms with specific antisera by DFA staining and procedures to detect nucleic acids of legionellae using PCR.

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The use of DFA to detect legionellae is limited by the number of specific antisera that can be used. Since there are no antisera which specifically react with all Legionella species, a different antiserum must be used for each species or serogroup. Reports on the sensitivity and specificity of DFA testing of environmental specimens vary greatly, with most studies indicating that the test is relatively insensitive and non-specific (Joly 1993). The use of PCR for detecting nucleic acids of legionellae in the environment has proved to be valuable in some investigations of outbreaks of legionellosis (Miller et al. 1993). A number of Legionella genes, including 5S rRNA, 16S rRNA, dotA, and mip genes, have been used as targets for PCR (Mahbubani et al. 1990; Starnbach et al. 1989; Yanez et al. 2005). Use of PCR to detect legionellae in the environment has indicated that up to 80% of freshwaters are positive while only 20–60% are positive by culture (Arnow et al. 1985; Fields et al. 2002; Flannery et al. 2006). Culture-independent techniques, particularly PCR, have indicated that some sites contain unusually high concentrations of legionellae when compared with culture results (Fliermans et al. 1981; Tison et al. 1983; Ortiz-Roque and Hazen 1987; Sheehan et al. 2005). PCR assays have also detected legionellae nucleic acids in environments considered unfavorable for the survival of these bacteria (Palmer et al. 1993). There are several potential explanations for the discrepancy between culturebased and culture-independent environmental analysis. First, a viable but nonculturable state (VNBC) has been described for L. pneumophila (Steinert et al. 1997). VNBC Legionella probably represent stressed bacteria. Bacteria stressed in water and wastewaters frequently become incapable of growth and colony formation under standard conditions because of structural or metabolic injury (Anonymous 1985). These bacteria can usually be cultivated through recovery enhancement procedures which provide essential nutrients or remove toxic or inhibitory compounds. For Legionella spp. the addition of host protozoa can reestablish the ability to culture the organism. Most environmental testing for legionellae is performed to assess the potential for disease transmission. If the differences in PCR and culture data are due to the presence of VNBC Legionella, the clinical relevance of these organisms must be established. Another potential explanation for the difference in culture and cultureindependent results is the presence of Legionella species which do not grow on laboratory media or are difficult to culture. These bacteria were originally described as Legionella-like amoebal pathogens or LLAPs and were detected by examining water samples for bacteria-infecting protozoa present in these samples (Rowbotham 1993; Adeleke et al. 1996). The number of new species of Legionella continues to grow and there may be a number of species of this organism which still cannot be cultured by conventional means. If these organisms account for the numerous PCR positive/culture negative samples, then their clinical relevance must be established as well. Finally, the differences in culture and culture-independent results may be the result of a non-specific reaction with unrelated organisms. As previously stated, a number of PCR and fluorescent antibody procedures for detecting legionellae in

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the environment have been described. Some of these assays were subject to limited or no validation procedures and may have resulted in unfounded conclusions regarding the distribution of legionellae in the environment. Until some of these issues are resolved, studies should not rely solely on culture-independent assays but should use these in conjunction with culture.

3. Natural Environments A limited number of studies have looked at the distribution of legionellae in natural waters. Most studies have focused on man-made aquatic systems associated with disease transmission. Several early studies of natural environments relied heavily on direct fluorescent antibody staining for the detection of the bacteria in these samples. One study examined hundreds of samples from numerous lakes and rivers in the US (Fleirmans et al. 1981). This study detected legionellae in the majority of samples using DFA but only 15% by isolation in guinea pigs. Natural freshwater environments are rarely associated with outbreaks of legionellosis. Most cases of legionellosis can be traced to man-made aquatic environments where the water temperature is higher than ambient temperature. At lower temperature, L. pneumophila multiplies more slowly in its protozoan host. This probably results in a natural balance between the bacteria and their protozoan host at concentrations below the threshold for human disease. Thermally altering aquatic environments can shift the balance between protozoa and bacteria, resulting in rapid multiplication of legionellae, which can translate into human disease. Legionella pneumophila multiplies at temperatures between 25 and 42°C with an optimal growth temperature of 35°C (Figure 5.1). The previously mentioned study of US lakes and streams detected a seasonal effect in one lake with higher rates of isolate in May, June, and July. Several studies have detected legionellae in naturally occurring thermal springs. A study of lakes and streams in Mount St. Helens blast zone in 1983 detected several species of legionellae with higher concentrations in water warmed by thermal vents (Tison et al. 1983). Studies in continental Portugal and the Azores identified multiple species of legionellae from waters ranging from Temperature Range of Legionellae Celsius

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Dormant

Growth

Death

FIGURE 5.1. Temperature Range of Legionellae.

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22 to 67.5°C (Verissimo et al. 1991). A PCR-based study of acidic biofilm communites in Yellowstone National Park detected several species of these bacteria in a pH 2.7 geothermal stream with temperatures ranging from 30to 38°C (Sheehan et al. 2005). These authors suggested that natural thermal gradients influence the Legionella species distribution. Legionellae have been detected in hot springs and associated resorts in multiple sites in Japan (Ohno et al. 2003). Many of these hot springs are modified for guest use and have incorporated the water treatment programs and recirculation and filtration systems. It is not clear if these legionellae populations naturally inhabit these enviroments with such distribution and concentration or if this is enhanced by the human manipulation of these waters. Although legionellae are realtively easy to detect in surface waters, the bacteria are believe to exist at low concentrations in ground water (Costas et al. 2005). The primary reason for this is believed to be the relatively colder temperatures of most ground water. However, a recent culture and PCR-based survey study found that 28–29% of ground waters tested were positive for legionellae with some of these positive ground waters as cold as 9°C (Brooks et al. 2004). Nevertheless, temperature appears to be the the primary environmental parameter associated with the prevalence of legioellae (Lee and West 1991; Flannery et al. 2006). Other factors such as osmolarity and pH appear to be less critical. High concentrations of sodium are known to be toxic to L. pneumophila and it is generally accepted that the bacteria cannot grow in sea water (Barbaree et al. 1983). Legionellae have not been cultured from sea water although one study did detect the organisms using PCR (Palmer et al. 1993). Conclusive studies are still needed to confirm the inhibition effect of sea water on the growth of Legionella. Although Legionella spp. are primarily associated with freshwater environments, the ecology of L. longbeachae appears to be a notable exception. This species is the leading cause of legionellosis in Australia which occurs in gardeners and those exposed to commercial potting soil (Steele 1989; Ruehlemann and Crawford 1996). The first US cases of L. longbeachae infection associated with potting soil were in 2000 (Centers for Disease Control and Prevention 2000). The factors that allow L. longbeachae to predominate in potting soil and thereby cause human disease are not yet understood.

4. Man-Made Environments Most of the information regarding the distribution of legionellae in man-made enviroments has been obtained during investigations of outbreaks of legionellosis. An overwhelming majority of cases of legionellosis can be traced to manmade aquatic environments where the water temperature is higher than ambient temperature. As stated in the 1992 Institute of Medicine report (Lederberg et al. 1992), “ Emerging Infections: Microbial Threats to Health in the United States,” “technology and industry can . . . cause, or at least contribute to, the emergence of infectious disease.” This is precisely the case with Legionnaires’ disease which may be regarded as a consequence of altering the environment for human benefit. A number of devices have been implicated as sources of aerosol transmission of


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TABLE 5.1. Building water systems known to transmit legionellae via aerosol. Type of water

Transmitting device

Potable

Showers, tap water faucets, respiratory therapy equipment Cooling towers and evaporative condensers, whirlpool spas, decorative fountains, ultrasonic mist machines, humidifiers

Non-potable

legionellae (Table 5.1). These sources are of two general types: those producing aerosols from potable water such as showers and faucets, and those from nonpotable water, such as cooling towers and whirlpool spas. Legionellae are common in many building water systems and only rarely cause disease. Prospective studies have found as many as 60% of buildings are colonized by these bacteria. A survey conducted in the UK found 60% of large buildings and 44% of cooling towers contained legionellae (Lee and West 1991). A study of buildings in San Francisco demonstrated that Legionella colonized 60% of hot water systems of 53 buildings tested over a two-year period (Flannery et al. 2006). Despite efforts to detect cases during the two-year study period, no case of Legionnaires’ disease were identified. Increased prevalence of Legionella colonization was associated with water heater temperatures below 50°C, buildings taller than 10 stories, and interruptions in water service. Approximately 30% of these buildings were consistently colonized over the two-year period. In most cases one or two strains or species of Legionella colonized a single building. In some cases a single strain or species was unique to a certain building. A similar study conducted in Pinellas County Florida detected Legionella in approximately 20% of the buildings tested (Moore et al. 2006). This demonstrates that the prevalence of Legionella varies in different geographic areas. The reason for this variablity is probably the result of many factors such as climate and water chemistry. The type of primary disinfectant used for municipal water supplies will effect the prevalence of legionellae in building water supplies. Monochloramine disinfection of water supplies is associated with decreased risk of Legionnaires’ disease (Kool et al. 1999; Heffelfinger et al. 2000). It is believed that monochloramine persists and penetrates biofilms better than free chlorine. Two prospective studies (mentioned above) looked at conversion from free chlorine to monochloramine as the primary disinfectant for the municipal water supply. In Pinellas County, Florida, the percent of buildings colonized with legionellae decreased from 19.8 to 6.2 % one month after conversion to monochloramine. In San Francisco County, Legionella colonization decreased from 60 to 4% after conversion and remained at this low level for the following year. Based on these studies the primary disinfectant used by a municipality should be a major factor in the prevalence of legionellae in man-made evironments. It is unclear if such treatment would have any effect of legionellae in non-potable systems such as cooling towers or whirlpool spas.

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5. Interaction with Other Organisms Legionellae require a distinct combination of nutrients in order to amplify in an environment. This unusual nutritional requirement of legionellae would appear to contradict the pervasiveness of the bacteria in aquatic environments. The levels of nutrients that legionellae require would rarely be found in freshwater and, if present, would serve only to amplify faster-growing bacteria that would compete with the legionellae. However, these nutrients represent an intracellular environment, not soluble nutrients commonly found in fresh water. Legionellae survive in aquatic and in some moist soil environments as intracellular parasites of freeliving protozoa (Rowbotham 1980; Fields 1996). Legionellae have been labeled “protozoonotic,” a term which appears to suit their natural history (Fields 1996). Legionellae have been reported to multiply in 14 species of amoebae, 2 species of ciliated protozoa, and 1 species of slime mold, while growth of legionellae in the absence of protozoa has been documented only on laboratory media (Fields 1996; Hägele et al. 2000; Solomon and Isberg 2000). Protozoa naturally present in environments implicated as sources of Legionnaires’ disease can support intracellular growth of legionellae in vitro (Barbaree et al. 1986). Legionella can infect and multiply intracellularly in both protozoa and human phagocytic cells (Horwitz and Silverstein 1980; Fields 1996). It appears that protozoa are the natural hosts of legionellae, whereas human phagocytic cells occasionally become ill-fated surrogates. Understanding the crucial role of protozoa in the ecology of legionellae is important for the development of successful prevention strategies.

6. Association with Biofilms Interaction of legionellae and protozoa is impacted by other microorganisms that comprise biofilms in building water systems. Legionellae are known to colonize these biofilms and can persist within these microbial communities for years. They are more easily detected from swab samples of biofilm than from flowing water, suggesting that the majority of the legionellae are biofilm associated (Rogers et al. 1994). A limited number of studies have attempted to characterize the bacteria’s interaction within these complex ecosystems. These studies have evaluated the effect of temperature and surface materials on the growth of L. pneumophila as well as the effect of biocides on sessile legionellae. The type of material used in the construction of plumbing systems has been shown to impact the ability of L. pneumophila to colonize these surfaces. L. pneumophila appears to be more abundant on elastomeric surfaces than copper or stainless steel (Rogers et al. 1994; Van der Kooij et al. 2005). However, the inhibitory effect of the metals is lost over time (Van der Kooij et al. 2005). The use of biofilm models to evaluate biocide efficacy against L. pneumophila represents a vast improvement over previous studies, which primarily evaluated the susceptibility of agar-grown bacteria in sterile water (Kusnetsov et al. 1994; Storey et al. 2004). These studies have


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also documented that amoebae provide protection from disinfectants in model biofilms (Donlan et al. 2005). The majority of Legionella/biofilm studies that have been conducted employ naturally occurring microbial communities. Such studies have the advantage of representing a true and natural microbial community, but all the organisms present have not been identified and their contribution to the survival and multiplication of legionellae remains unknown. Biofilm matrices are known to provide shelter and a gradient of nutrients. The complex nutrients available with biofilms have led some researchers to propose that the biofilms support the survival and multiplication of legionellae outside a host cell (Rogers and Keevil 1992). This concept is certainly plausible; most facultative intracellular bacteria are known to multiply extracellularly in some environments. If legionellae can multiply extracellularly within biofilms, the characterization of this phenomenon would have impact on control strategies for the prevention of legionellosis. Investigators have attempted to detect extracellular growth of L. pneumophila using a biofilm reactor and a defined bacterial biofilm grown on non-supplemented potable water (Murga et al. 2001). The base biofilm was composed of Pseudomonas aeruginosa, Klebsiella pneumoniae, and the Flavobacterium-like organism isolated from a water sample containing legionellae. The addition of the amoeba Hartmannella vermiformis to the reactor resulted in a reproducible equilibrium between the amoeba and heterotrophic bacteria. L. pneumophila associated with, and persisted in, these biofilms with and without H. vermiformis. L. pneumophila cells did not appear to develop microcolonies, and growth measurement studies indicate that L. pneumophila did not multiply within this biofilm in the absence of amoebae. L. pneumophila did multiply in the biofilm and planktonic phase in the presence of H. vermiformis, and the majority of these bacteria appeared to be shed into the planktonic phase. These studies suggest that L. pneumophila may persist in biofilms in the absence of amoebae, but in the model, the amoebae were required for multiplication of the bacteria. This model biofilm was constructed of five preselected organisms and does not represent the many potentially diverse biofilms that may support the growth of legionellae in the environment. Additional studies are needed to determine if legionellae possess a means to multiply independent of a host cell within biofilms. The control of biofilm-associated legionellae may lead to the most effective control measures to prevent legionellosis. Institutions that have experienced outbreaks of legionellosis are all too aware of how tenacious legionellae can be within building water system biofilms.

References Adeleke A, Pruckler J, Benson R, Rowbotham T, Halablab M, Fields B (1996) Legionellalike amebal pathogens—phylogenetic status and possible role in respiratory disease. Emerg Infect Dis 2:225–230. Anonymous (1985) Stress organisms. In: Franson MAH, Clesceri LS, Greenberg AE, Trussell RR, (eds.) Standard methods for the examination of water and wastewater, 16th edition. American Public Health Association. Washington, DC. pp. 1036–1038.

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Arnow PM, Weil D, Para MF (1985) Prevalence and significance of Legionella pneumophila contamination of residential hot-tap water systems. J Infect Dis 152:145–151. Barbaree JM, Sanchez A, Sanden GN (1983) Tolerance of Legionella species to sodium chloride. Curr Microbiol 9:1–5. Barbaree JM, Fields BS, Feeley JC, Gorman GW, Martin WT (1986) Isolation of protozoa from water associated with a legionellosis outbreak and demonstration of intracellular multiplication of Legionella pneumophila. Appl Environ Microbiol 51:422–424. Brooks T, Osicki RA, Springthrorpe VS, Sattar SA, Filion L, Abrial D, Riffard S (2004) Detection and identification of Legionella species from ground waters. J Toxicol Environ Health, Part A 67:1845–1859. Centers for Disease Control and Prevention. (2000) Legionnaires’ Disease associated with potting soil—California, Oregon, and Washington, May–June 2000. MMWR 49(34):777–778. Costas J, Tiago I, da Costa MS, Verissiom A. (2005) Presence and persistence of Legionella spp in groundwater. Appl Environ Microbiol 71:663–671. Donlan RM, Forster T, Murga R, Brown E, Lucas C, Carpenter J, Fields B (2005) Legionella pneumophila associated with the protozoan Hartmannella vermiformis in a model multispecies biofilm has reduced susceptibility to disinfectants. Biofouling 21: 1–7. Fields BS (1996) The molecular ecology of Legionellae. Trends Microbiol 4:286–290. Fields BS, Benson RF, Besser RE (2002) Legionella and Legionnaires’ Disease: 25 years of investigation. Clin Microbiol Rev 15:506–526. Flannery B, Gelling LB, Vugia DJ, Weintraub JM, Salerno JJ, Conroy MJ, Stevens VA, Rose CE, Moore MR, Fields BS, and Besser RE (2006) Reduced Legionella after monochloramine disinfection. Emerg Infect Dis. 12:588–596. Fliermans CB, Cherry WB, Orrison LH, Smith SJ, Tison DL, Pope DH (1981) Ecological distribution of Legionella pneumophila. Appl Environ Microbiol 41:9–16. Green PN, Pirrie RS (1993) A laboratory apparatus for the generation and biocide efficacy testing of Legionella biofilms. J Appl Bacteriol 74:388–393. Hägele S, Kohler R, Merkert H, Schleicher M, Hacker J, Steinert M (2000) Dictyostelium discoideum: a new host model system for intracellular pathogens of the genus Legionella. Cell Microbiol 2:165–171. Heffelfinger JD, Kool JL, Fridkin SK, Fraser VJ, Hageman J, Carpenter, J, Whitney CG; Society for Healthcare Epidemiology of America (2000) Risk of hospital-acquired legionnaires’ disease in cities using monochloramine versus other water disinfectants. Infect Control Hops Epidemiol 24:569–574. Horwitz MA, Silverstein SC (1980) The legionnaires’ disease bacterium (Legionella pneumophila) multiplies intracellularly in human monocytes. J Clin Inves 66: 441–450. Joly JR (1993) Monitoring for the presence of Legionella: where, when, and how? In: Barbaree JM, Breiman RF, Dufour AP (eds.). Legionella: Current status and emerging perspectives. American Society for Microbiology, Washington, DC, pp. 211–216. Katz SM, Hammel, JM (1987) The effect of drying, heat, and pH on the survival of Legionella pneumophila. Ann Clin Lab Sci 17:150–156. Kool JL, Carpenter JC, Fields BS (1999) Effect of monochloramine disinfection of municipal drinking water on risk of nosocomial Legionnaires’ disease. Lancet 353:272–277. Kusnetsov JM, Keskitalo PJ, Ahonen HE, Tulkki AI, Miettinen IT, Martikanen PJ (1994) Growth of Legionella and other heterotrophic bacteria in a circulating cooling water system exposed to ultraviolet irradiation. J Appl Bacteriol 77:461–466. Lederberg J, Shoppe RE,Oaks SCJr.(eds.). (1992) Emerging Infections: Microbial Threats to health in the United States. National Academy Press, Washington, DC


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Lee JV, West AA (1991) Survival and growth of Legionella species in the environment. J Appl Bacteriol Symp Supp 70:121–129. Mahbubani MH, Bej AK, Miller R, Haff L, DiCesare J, Atlas RM (1990) Detection of Legionella pneumophila with polymerase chain reaction and gene probe methods. Mol Cell Probes 4:175–187. Mermel LA, Joesephson SL, Giorgio CH, Dempsey J, Parenteau S (1995) Association of Legionnaires’ disease with construction: contamination of potable water? Infect Contr Hosp Epidemiol 16:76–80. Miller LA, Beebe JL, Butler JC, Martin WT, Benson R, Hoffman RE, Fields BS (1993) Use of polymerase chain reaction in an epidemic investigation of Pontiac fever. J Infect Dis 168:769–772. Moore MR, Pryor M, Fields B, Lucas C, Phelan M, Besser Re (2006) Introduction of monochloramine into a municipal water system: impact on colonization of buildings by Legionella. Appl Environ Microbiol. 72:373–383. Murga R, Forster TS, Brown E, Pruckler JM, Fields BS, Donlan RM (2001) The role of biofilms in the survival of Legionella pneumophila in a model potable water system. Microbiology 147:3121–3126. Ohno A, Kato N, Yamada K, Yamaguchi K (2003) Factors influencing survival of Legionella pneumophila serotype 1 in hot spring water and tap water. Appl Environ Microbiol 69:2540–2547. Ortiz-Roque CM, Hazen TC (1987) Abundance and distribution of Legionellaceae in Puerto Rican waters. Appl Environ Microbiol 53:2231–2236. Palmer CJ, Tsai YL, Paszko-Kolva C, Sangermano LR (1993) Detection of Legionella species in sewage and ocean water by polymerase chain reaction, direct fluorescentantibody, and plate culture methods. Appl Environ Microbiol 59:3618–3624. Rogers J, Keevil CW. 1992. Immunogold and fluorescein immunolabelling of Legionella pneumophila within an aquatic biofilm visualized by using episcopic differential interference contrast microscopy. Appl Environ Microbiol 58:2326–2330. Rogers JA, Dowsett B, Dennis PJ, Lee JV, Keevil CW (1994) Influence of temperature and plumbing material selection on biofilm formation and growth of Legionella pneumophila in a model potable water system containing complex microbial flora. Appl Environ Microbiol 60:1585–1592. Rowbotham TJ (1980) Preliminary report on the pathogenicity of Legionella pneumophila for freshwater and soil amoebae. J Clin Pathol 33:1179–1183. Rowbotham TJ (1993) Legionella-like amoebal pathogens. In: Barbaree JM, Breiman RF, Dufour AP (eds.). Legionella: Current status and emerging perspectives. American Society for Microbiology, Washington, DC pp. 137–140. Ruehlemann SA, Crawford GR (1996) Panic in the potting shed: The association between Legionella longbeachae serogroup 1 and potting soils in Australia. Med J Aust 164:36–38. Sheehan KB, Henson JM, Ferris MJ. (2005) Legionella species diversity in an acidic biofilm community in Yellowstone National Park. Appl Environ Microbiol 71:507–511. Solomon JM, Isberg RR (2000) Growth of Legionella pneumophila in Dictyostelium discoideum: a novel system for genetic analysis of host-pathogen interactions. Trends Microbiol 8:478–480. Starnbach MN, Falkow S, Tompkins LS (1989) Species-specific detection of Legionella pneumophila in water by DNA amplification and hybridization. J Clin Microbiol 27:1257–1261. Steele TW (1989) Legionnaires’ disease in South Australia, 1979–1988. Med J Aust 151:322, 325–326, 328.

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Steinert M, Emody L, Amann R, Hacker J (1997) Resuscitation of viable but nonculturable Legionella pneumophila JR32 by Acanthamoeba castellanii. Appl Environ Microbiol 63:2047–2053. Storey MV, Langmark J, Ashbolt NJ, Stenstrom TA (2004) The fate of legionellae within distribution pipe biofilms: Measurement of their persistence, inactivation, and detachment. Water Sci Tech 49:269–275. Tison DL, Baros JA, Seidler RJ (1983) Legionella in aquatic habitats in the mount Saint Helens blast zone. Curr Microbiol 9:345–348. Van der Kooij D, Veenendaal HR, Scheffer WJH (2005) Biofilm formation and multiplication of Legionella in a model warm water system with pipes of copper, stainless steel and cross-linked polyethylene. Water Res 39:2789–2798. Verissimo A, Marrao G, Gomes da Silva F, da Costa MS (1991) Distribution of Legionella spp. in hydrothermal areas in continental Portugal and the island of Sao Miguel, Azores. Appl Environ Microbiol 57:2921–2927. Yanez MA, Carrasco-Serrano C, Barbera VM, Catalan V (2005) Quantitative detection of Legionella pneumophila in water samples by immunomagnetic purification and realtime PCR amplification of the dotA gene. Appl Environ Microbiol 71:3433–3441.

6 Regulation of the Legionella pneumophila Life Cycle Rachel L. Edwards and Michele S. Swanson

1. Introduction Legionella pneumophila, the gram-negative intracellular pathogen responsible for the potentially fatal pneumonia known as Legionnaires’ Disease, likely resides in complex biofilms in both natural and man-made water systems (Fields et al. 2002). However, when L. pneumophila are confronted by various freshwater protozoa they can efficiently establish an intracellular niche protected from digestion, including within 14 species of amoebae, two species of ciliated protozoa, and one species of slime mold (Fields et al. 2002). Pioneering studies by Rowbotham demonstrated that L. pneumophila alternates between two distinct phenotypic states in amoebae: a non-motile, thin-walled replicative form that contains little ␤-hydroxybutyrate and a motile, thick-walled infectious form that contains the ␤-hydroxybutyrate storage granules (Rowbotham 1986; Garduño et al. 2002). With advances in molecular genetics and microscopy, many of the early findings regarding the L. pneumophila life cycle have been confirmed, and insight to the diversity and versatility of the bacterium has been obtained. Here we summarize both the developmental biology of L. pneumophila that is governed by nutrient supply as well as the regulatory components controlling its differentiation.

2. Life Cycle of L. pneumophila When planktonic, transmissive L. pneumophila are engulfed by phagocytic cells, the bacteria avoid lysosomal degradation and instead establish vacuoles isolated from the endocytic network (Fig. 6.1). To gauge nutrient conditions within its host cell, L. pneumophila employs the Pht family of transporters (Sauer et al. 2005). If vacuolar conditions are favorable, the post-transcriptional regulator CsrA, and perhaps the sRNA chaperone Hfq, suppress transmissive traits and promote intracellular replication (Fig. 6.2) (Fettes et al. 2001; Molofsky and Swanson 2003; McNealy et al. 2005). When nutrients are depleted, bacterial replication halts and the ribosomal enzyme RelA produces (p)ppGpp (Hammer and Swanson 1999; Zusman et al. 2002). The accumulation of (p)ppGpp in the bacterial cytosol either 95

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FIGURE 6.1. Life cycle of L. pneumophila. (1) Transmissive L. pneumophila engulfed by phagocytic cells reside in vacuoles and avoid lysosomal degradation. (2) Under favorable conditions, transmissive bacteria begin to replicate. (3) When nutrients are depleted, replicating bacteria stop dividing and begin to express transmission traits. (4) Microbes may develop into a more resilient and infectious mature intracellular form (MIF). (5) The host cell is lysed and transmissive microbes are released into the environment. (6) L. pneumophila that do not encounter a new host cell probably establish biofilms in water systems and ponds. (7) When microbes encounter a host cell, the cycle begins anew. (8) L. pneumophila cultured in broth to either exponential or stationary phase exhibit many of the traits of the replicative and transmissive forms, respectively. Modified from Molofsky AB and Swanson MS (2004) Mol Microbiol 53(1):29–40.

directly or indirectly stimulates the LetA/LetS two-component system to relieve CsrA repression of transmissive traits (Hammer and Swanson 1999; Hammer et al. 2002; Molofsky and Swanson 2003). Together, the LetA/LetS system, the enhancer protein LetE, and the alternative sigma factors RpoS, RpoN, and FliA induce traits thought to promote efficient host transmission and survival in the environment, including evasion of phagosome–lysosome fusion, motility, cytotoxicity, sodium sensitivity and resistance to environmental stresses (Bachman and Swanson 2001; Hammer et al. 2002; Lynch et al. 2003; Bachman and Swanson 2004a; Bachman and Swanson 2004b; Jacobi et al. 2004). Under particular conditions, L. pneumophila further develops into the highly resilient and infectious cell type, the mature intracellular form (MIF) (Faulkner and Garduño 2002; Garduño et al. 2002). Eventually, the exhausted host cell lyses, and progeny are released into the environment. While L. pneumophila that fail to find a new phagocyte probably establish complex biofilms, planktonic bacteria that encounter another suitable host can initiate the intracellular life cycle once more.

6. Regulation of the Legionella pneumophila Life Cycle

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FIGURE 6.2. A model for regulation of L. pneumophila differentiation. Arrows indicate activation and bars indicate inhibition. Replicative phase regulatory interactions are represented by solid double lines, while transmission phase regulatory pathways are indicated by a single solid line. Speculative interactions are designated by dotted lines.

3. Broth Model Broth cultures of L. pneumophila grown to either the exponential or stationary phase exhibit traits similar to replicative and transmissive bacteria, respectively, that are observed in co-cultures with phagocytic cells (Fig. 6.1). While many of the different stages and regulatory elements of the L. pneumophila life cycle were originally discerned by observing synchronous broth cultures, subsequent analysis in eukaryotic cells has supported many of these findings. Likewise, comparison of the transcription profiles of L. pneumophila cultured in broth and in Acanthamoeba castellanii has revealed that 84% of replicative phase genes and 77% of transmissive phase genes are upregulated both in vitro and in vivo, thereby confirming the utility of broth culture studies (Brüggemann et al. 2006). Several lines of evidence suggest that the replicative and transmissive phases observed in both broth and eukaryotic cell cultures are reciprocal. For example, when stationary phase L. pneumophila are cultured with eukaryotic cells, they suppress their transmissive traits of cytotoxicity, sodium sensitivity, and motility, and instead replicate profusely (Byrne and Swanson 1998; Alli et al. 2000). Following the replicative period, transmissive traits are induced, and the host cell lyses (Byrne and Swanson 1998; Alli et al. 2000). Similarly, FlaA, Mip, DotH, and DotO proteins, which are known to enhance invasion of eukaryotic cells, are expressed during the entry and exit periods, but not during replication

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(Hammer and Swanson 1999; Watarai et al. 2001; Wieland et al. 2002). In contrast, the promoter of CsrA, a repressor of transmission traits, is only active during the replicative period, not during invasion or host cell lysis (Molofsky and Swanson 2003). While pure bacterial cultures are advantageous for many molecular and biochemical techniques, several characteristics of L. pneumophila have only been observed in vivo, emphasizing the simplicity and limitations of the broth model. For instance, the replication vacuole in A/J mouse macrophages acidifies and L. pneumophila remain acid tolerant, whereas exponentially growing broth cultures are acid sensitive (Sturgill-Koszycki and Swanson 2000). Additionally, bacteria harvested from A. castellanii are more infectious than agar-grown bacteria, suggesting that the intracellular environment of amoebae can affect virulence traits (Cirillo et al. 1999). Moreover, after extended culture in HeLa cells, L. pneumophila differentiates to the cyst-like MIF, a cell type also observed in amoebae and clinical samples, but not in broth culture (Greub and Raoult 2003; Garduño et al. 2002). The substantial differences observed between broth and phagocytic cell cultures highlight the impact of experimental design and lend caution to making inferences based on any single laboratory model.

4. Amino Acid Availability Governs Differentiation Although the exact nutrient composition of the L. pneumophila replication vacuole is unknown, several lines of evidence indicate that amino acids are critical, and differences in these concentrations can affect the differentiation state of the microbe. Foremost, broth studies indicate that L. pneumophila depends on amino acids for its sole source of carbon and energy (Tesh et al. 1983). Additionally, the uptake of amino acids by its host cell via the human transporter protein SLC1A5 (hATB0,⫹) is required for L. pneumophila to replicate in macrophages (Wieland et al. 2005). Furthermore, L. pneumophila uses amino acid transporters to determine the nutrient availability of the environment and trigger its differentiation as deemed appropriate (discussed below) (Sauer et al. 2005). Finally, when amino acids are depleted, L. pneumophila utilizes the stringent response to induce a panel of traits that enable escape from its spent host, survival in the environment, and the capacity to invade another suitable host (discussed below) (Hammer and Swanson 1999). The regulatory linkage of nutrient availability to differentiation is essential for L. pneumophila pathogenesis as it determines the phenotypic profile of the microbe.

4.1. Pht Family of Transporters To sense amino acid availability and determine if differentiation to a replicative state is advantageous, transmissive L. pneumophila use a family of phagosomal transporters (Phts) (Sauer et al. 2005). In particular, a L. pneumophila mutant of the phtA gene was shown to have a pronounced defect in intracellular growth in


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FIGURE 6.3. Pht transporters couple nutrient acquisition to microbial differentiation. Transport of amino acids by Pht proteins trigger intracellular transmissive L. pneumophila to differentiate to the replicative form. Amino acid acquisition via Pht proteins is also essential for L. pneumophila to replicate in macrophages. When replicative bacteria fail to acquire essential amino acids, L. pneumophila induce expression of transmission traits.

murine bone marrow-derived macrophages, and it failed to differentiate to the replicative state (Sauer et al. 2005). However, the ability of the phtA mutant to infect macrophages and establish a protected ER-derived vacuole was not compromised (Sauer et al. 2005). Using sequence analysis, PhtA was predicted to be a transmembrane protein that traverses the membrane 12 times and belong to the Major Facilitator Superfamily of transporters (Pao et al. 1998; Sauer et al. 2005). Indeed, the growth defect of the phtA mutant in both minimal media and macrophages can be bypassed by supplementing media with free threonine or threonine dipeptides (Sauer et al. 2005). Taken together with the aforementioned data, it is proposed that L. pneumophila uses the PhtA transporter as a mechanism to couple nutrient acquisition to differentiation (Figure 6.3) (Sauer et al. 2005). Examination of the L. pneumophila genome reveals that strains Philadelphia 1 and Lens have 11 phtA homologs, while the Paris strain has 9 (Cazalet et al. 2004; Sauer et al. 2005). Though several of these loci are likely gene duplications, it is conceivable that L. pneumophila encodes pht homologs dedicated to transporting each of the six essential amino acids, either arginine, cysteine, methionine, serine, threonine, or valine (Ristroph et al. 1981). In support of this model, a phtJ (previously milA) mutant has a growth defect in macrophages and alveolar epithelial cells (Harb and Abu Kwaik 2000), which can be suppressed by valine supplementation (Sauer and Swanson, unpublished). Other vacuolar pathogens may also exploit transporters to evaluate nutrient availability before undergoing differentiation, since pht homologs have been identified in Coxiella burnetti and Francisella tularensis, two closely related pathogens that also efficiently parasitize both

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macrophages and amoebae (Oyston et al. 2004). Thus, the Pht family of transporters may equip vacuolar pathogens to assess the nutrient composition in their protective vacuole before committing to differentiation.

4.2. Stringent Response Microbes trigger a global change in their cellular metabolism known as the stringent response when confronted with nutritional and metabolic stresses, such as the limitation of amino acids, carbon, nitrogen, or phosphate (Magnusson et al. 2005). Numerous physiological processes are coordinately altered, including growth inhibition, down-regulation of nucleic acid and protein synthesis, enhancement of protein degradation, and upregulation of amino acid synthesis and transport. The stringent response is triggered when uncharged tRNAs accumulate in the A-site of the 50S ribosome. Using ATP as a phosphate donor, the ribosome-associated enzyme RelA is activated to synthesize the alarmone, ppGpp (guanosine 3⬘,5⬘-bispyrophsophate), and its precursor, pppGpp (guanosine 3⬘-diphosphate, 5⬘-triphosphate), by phosphorylating GDP and GTP, respectively. The ppGpp effector molecule then binds directly to the ␤ and ␤⬘ subunits of the RNA polymerase (RNAP) core enzyme. As a result, the transcription of some classes of genes is upregulated, while the expression of other sets of genes is inhibited (Magnusson et al. 2005). There is considerable evidence that (p)ppGpp acts as a global regulator to modulate a variety of bacterial cellular and physiological processes. Furthermore, numerous intracellular and extracellular pathogens appear to exploit the stringent response pathway to activate virulence genes and persist in the hostile environment of their host; these include Mycobacterium tuberculosis, Listeria monocytogenes, Pseudomonas aeruginosa, Streptococcus pyogenes, Staphylococcus aureus, Borrelia burgdorferi, and Salmonella typhimurium (Godfrey et al. 2002; Magnusson et al. 2005). By monitoring environmental conditions and invoking the stringent response, pathogens can elicit swift changes in gene expression to adapt to the metabolic stresses encountered in their host, thereby promoting selfpreservation. Legionella pneumophila accumulate (p)ppGpp upon entry into stationary phase, when amino acids are depleted, or when relA is induced (Hammer and Swanson 1999). As a result, the microbe converts from a replicative to transmissive state, as characterized by the expression of virulence traits (Hammer and Swanson 1999). L. pneumophila relA mutants are unable to produce detectable levels of (p)ppGpp and are defective for the transmission traits of pigmentation and motility (Zusman et al. 2002). However, relA is dispensable for intracellular growth in both human macrophages and amoebae, suggesting relA activity contributes exclusively to the transmissive phase and is not required for intracellular replication (Zusman et al. 2002). By analogy to other bacteria that use a stringent response, it is proposed that (p)ppGpp acts as an alarmone to induce the coordinate expression of traits that equip L. pneumophila to escape its spent host and persist in the environment until it encounters another phagocyte.


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In L. pneumophila, RelA appears to be the major (p)ppGpp synthase because relA mutants do not accumulate detectable levels of the alarmone (Zusman et al. 2002). However, many prokaryotes contain a second enzyme, SpoT, which, depending on environmental conditions, exhibits either (p)ppGpp synthase or hydrolase activity (Chatterji and Kumar Ojha 2001). When growth conditions are favorable, SpoT works together with RelA to maintain stable levels of (p)ppGpp in the cytosol through its hydrolase activity (Chatterji and Kumar Ojha 2001); but, under certain stresses and nutrient limitations except amino acid starvation, SpoT can synthesize (p)ppGpp (Magnusson et al. 2005). Sequence analysis indicates that the L. pneumophila genome contains a homolog 52% identical to the Escherichia coli spoT gene that appears to be essential for viability, as attempts to delete the locus were unsuccessful (Zusman et al. 2002). Further analysis can determine whether SpoT contributes to an additional mechanism by which L. pneumophila respond to external stress to make decisions regarding its fate.

5. Evidence that Other Factors Trigger Differentiation Although the stringent response is the most well-characterized mechanism by which L. pneumophila controls differentiation, several lines of evidence indicate that other signals are likely to be involved. By phenotypic analysis, relA mutants appear less defective for transmissive traits than other known regulators of differentiation, suggesting additional factors may act in concert with RelA (Hammer and Swanson 1999; Zusman et al. 2002). Also, letA mutants (discussed below) can express traits that are normally impaired if certain growth conditions are altered, such as aeration, temperature, or media composition (Fernandez-Moreia and Swanson, unpublished). Furthermore, broth cultures of letA and letS mutants are defective in their ability to infect mouse macrophages, but surviving bacteria can initiate secondary and tertiary infections as efficiently as wild-type (Hammer et al. 2002). Additionally, L. pneumophila letA mutants are more infectious when harvested from cultures of bone marrow macrophages (Byrne and Swanson, unpublished). Taken together, these data indicate that additional regulators can bypass RelA and the LetA/LetS two-component system by receiving signals and initiating differentiation. Whether the signals stem from the unique environment of the L. pneumophila vacuole or are only induced when bacterial density within the vacuole reaches a certain threshold remains to be determined.

5.1. Acetyl-Phosphate In addition to (p)ppGpp, acetyl-phosphate can act as a global signal for many cellular processes, including chemotaxis, nitrogen and phosphate assimilation, biofilm development, and the expression of virulence traits (Wolfe 2005). Acetylphosphate is a high energy phosphate compound that can be synthesized by two separate reactions: (1) from acetyl-CoA and Pi by phosphotransacetylase, encoded by the pta gene, or (2) from ATP and acetate, in a reaction catalyzed by

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acetyl kinase, the product of ackA (McCleary et al. 1993; Wolfe 2005). Response regulators of two-component systems can use acetyl-phosphate to catalyze their own phosphorylation (Wolfe 2005). Accordingly, the Bordetella pertussis response regulator, BvgA, can be phosphorylated in the absence of its cognate histidine kinase using acetyl-phosphate as a donor (Boucher et al. 1994). Likewise, either acetyl phosphate or the histidine kinase BarA can activate the S. typhimurium response regulator SirA to induce the expression of invasion genes (Lawhon et al. 2002). Indeed, preliminary data indicate that acetate can trigger replicating L. pneumophila to express several transmission phase phenotypes (Edwards and Swanson, unpublished). The L. pneumophila genome contains homologs of both pta and ackA of E. coli (Gal-Mor and Segal 2003b), suggesting acetyl-phosphate may provide a signal for the induction of genes unique to the transmissive phase, and thus trigger bacterial differentiation.

6. Transcriptional Control of Differentiation via Sigma Factors To regulate gene expression, one mechanism commonly employed by bacteria is to modify RNA polymerase (RNAP) activity by altering its sigma factor. In E. coli, RNAP consists of a core enzyme and one of seven potential sigma factors that direct the RNAP to a distinct cohort of promoters (Nystrom 2004). For growth-related activities and proliferation, E. coli require the housekeeping sigma factor, ␴70, encoded by rpoD (Nystrom 2004). However, during conditions of growth arrest, starvation, stress, or maintenance, the bacteria replace ␴70 with alternate sigma factors, thereby recruiting RNAP to the promoters of a cohort of genes that will confer survival under the deleterious conditions (Nystrom 2004). Analysis of the L. pneumophila genome has identified six alternative sigma factors: RpoD (␴D/␴70), RpoE, RpoH, RpoN (␴54), RpoS (␴S/␴38), and FliA (␴28), as well as the sigma factor-dependent enhancer, FleQ, and the anti-sigma factors, FlgM and anti-sigma factor B (Cazalet et al. 2004; Chien et al. 2004). Several of the L. pneumophila sigma factors have been implicated by genetic analysis to regulate subsets of transmission traits (Molofsky et al. 2005; Bachman et al. 2001). Recent biochemical and genetic data indicate that the effector molecule of the stringent response, (p)ppGpp, controls sigma factor competition for the RNAP core enzyme (Nystrom 2004). In particular, (p)ppGpp not only regulates the ability of different alternative sigma factors to bind RNAP but also controls the production and activity of many sigma factors (Magnusson et al. 2005). Therefore, under nutrient-limiting conditions, the stringent response governs sigma factor competition and other aspects of gene transcription, thus altering the expression profile and enhancing the fitness of the microbe.


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6.1. RpoS The stationary phase sigma factor, RpoS, is required for sodium sensitivity, maximal expression of flagellin, and lysosomal evasion, but it is dispensable for the other known transmissive phase phenotypes (Hales and Shuman 1999; Bachman and Swanson 2001). In accordance with the theory of sigma factor competition, over-expression of rpoS decreases csrA, letE, fliA, and flaA transcripts, and inhibits the fliA-dependent transmission traits of motility, infectivity, and cytotoxicity (Bachman and Swanson 2004a). Also, multiple copies of rpoS inhibit intracellular replication in A. castellanii (Hales and Shuman 1999). Although the global regulation of gene expression by sigma factors in L. pneumophila has not been confirmed biochemically, these data support the model that during stringency, (p)ppGpp alters the competition among sigma factors for RNAP, thus allowing recruitment of RNAP to the appropriate set of transmission genes (Hales and Shuman 1999; Bachman and Swanson 2004a).

6.2. RpoN and FleQ Since the assembly of the bacterial flagellum is an energetically taxing process, microbes encode a complex regulatory cascade to ensure that the timing and synthesis of both structural and accessory proteins of the flagellar regulon are tightly controlled. In L. pneumophila, the alternative sigma factor RpoN and the coactivator FleQ are at the top of this transcriptional hierarchy; whereby rpoN and fleQ mutants lack a flagellum and produce very little flagellin protein (Jacobi et al. 2004). Additionally, both RpoN and FleQ positively regulate transcription of several genes within the second tier of the regulation cascade, including fliM, fleN, and fleSR (Jacobi et al. 2004). In contrast, RpoN and FleQ are dispensable for fliA and flaA transcription, suggesting that additional factors may govern genes within the transcriptional cascade of the L. pneumophila flagellar regulon (Jacobi et al. 2004).

6.3. FliA To induce flaA transcription, assemble a monopolar flagellum, and become motile, L. pneumophila require a second sigma factor, FliA (Heuner et al. 1997, 2002). Besides controlling genes of the flagellar regulon, FliA governs a set of motility-independent traits thought to promote host transmission and persistence. In particular, L. pneumophila require FliA to produce a melanin-like pigment, alter its surface properties, avoid lysosomes, and replicate within Dictyostelium discoideum (Hammer et al. 2002; Heuner et al. 2002; Molofsky et al. 2005). By coordinating motility with other essential virulence traits, L. pneumophila can fine-tune its expression profile to enhance its versatility when confronted by a multitude of host defenses and environmental stresses.

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7. Post-Transcriptional Control of Differentiation 7.1. LetA/LetS Two-Component System Two-component systems are widely used by prokaryotic organisms to adapt to environmental fluctuations. Typically, these signal transduction systems consist of a membrane-bound sensor protein that monitors the environment, and a cytoplasmic response regulator that binds target DNA sequences (Appleby et al. 1996; Bijlsma and Groisman 2003). Upon stimulation, the sensor autophosphorylates a conserved histidine residue using the ␥-phosphoryl group of ATP as a donor (Appleby et al. 1996; Bijlsma and Groisman 2003). The phosphate is then transferred to an aspartic acid in the response regulator, allowing for activation or repression of target genes (Appleby et al. 1996; Bijlsma and Groisman 2003). The specific signal for autophosphorylation may be abiotic or biotic, and be produced by the environment, the host cell, or generated by the bacteria themselves via quorum sensing (Heeb and Haas 2001). However, for many microbial two-component systems, the appropriate signal remains elusive. The LetA/LetS (Legionella transmission activator and sensor, respectively) system of L. pneumophila was originally identified by screening mutants defective for flagellin expression (Hammer et al. 2002). Further analysis demonstrated that, as cells exit exponential phase, LetA/LetS induces an array of traits likely to promote transmission, including the ability to infect both macrophages and A. castellanii, avoid phagosome–lysosome fusion, sodium sensitivity, stress resistance, motility, pigmentation, and macrophage cytotoxicity (Figure 6.2) (Hammer et al. 2002; Lynch et al. 2003; Bachman and Swanson 2004b). Accordingly, letA and letS mutants do not express transmissive traits, and instead constitutively display phenotypes similar to wild-type replicative bacteria (Hammer et al. 2002; Lynch et al. 2003). Bacteria that lack LetA fail to respond to the alarmone (Hammer et al. 2002), but whether LetS is activated directly by (p)ppGpp or another signaling molecule can stimulate its autophosphorylation is unknown. But, by analogy to other two-component systems, LetS is the proposed membrane-associated sensor kinase and LetA, the cognate response regulator. LetA/LetS belongs to a family of signal-transducing proteins that includes BvgA/BvgS of Bordetella, SirA/BarA of Salmonella, GacA/GacS of Pseudomonas, and the UvrY/BarA, EvgA/EvgS, and TorR/TorS systems of E. coli (reviewed by Perraud et al. 1999; Heeb and Haas 2001). The BvgA/BvgS system, which established the paradigm for this family of signaling molecules, deviates from classic two-component systems by employing a four-step phosphorelay in which His-Asp-His-Asp residues are sequentially phosphorylated (Uhl and Miller 1996). BvgS is a tripartate, transmembrane sensor whose periplasmic domain is linked by a membrane-spanning region to three cytoplasmic signaling domains: transmitter, receiver, and histidine phosphotransfer domain (Cotter and DiRita 2000). In response to an unknown signal, BvgS autophosphorylates a histidine in the transmitter, and then sequentially transfers the phosphoryl group to aspartic acid and histidine residues in the receiver and the histidine phosphotransfer


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domain, respectively (Cotter and DiRita 2000). BvgA is the response regulator that, when transphosphorylated by BvgS, gains affinity for Bvg-activated promoters (Cotter and DiRita 2000). It is suggested that the multi-step design of this family of two-component systems enables the bacteria to express a spectrum of phenotypes in response to different environmental conditions. In support of this model, Bordetella alternate between several distinct phenotypic phases, and it is the BvgA/BvgS system that regulates the phases by temporally controlling the expression of different classes of genes (Cotter and Miller 1997). Similar to BvgA/BvgS, the L. pneumophila LetA/LetS two-component system likely utilizes a four-step phosphorelay to fine-tune its panel of transmission traits. A threonine-to-methionine substitution, four residues from the proposed autophosphorylation site in LetS, creates a L. pneumophila mutant locked in a Let-intermediate (leti) phase (Edwards and Swanson, unpublished). When compared with wild-type and letS null bacteria, leti mutants are intermediate in their ability to enter and survive in macrophages and intermediately cytotoxic. In contrast, leti mutants resemble either letS null bacteria or wild-type cells for each of the other known transmissive phase phenotypes (Edwards and Swanson, unpublished). Interestingly, while only 30–50% of leti mutants are motile, promoter analysis of the flaA gene demonstrates that the entire population of cells express flaA, though later than wild-type, indicating that robust control of the flagellar regulon is required for flagellum assembly (Edwards and Swanson, unpublished). Together these results support the hypothesis that L. pneumophila may use the multi-step design of the LetA/LetS two-component system to customize its array of traits, thereby enhancing its versatility and fitness. All transmission traits activated by LetA/LetS and repressed by CsrA (discussed below) are also affected by a mutation in letE, suggesting that LetE either enhances LetA/LetS function or inhibits CsrA activity (Bachman and Swanson 2004b). LetE apparently functions as a small protein, rather than a regulatory RNA, but the mechanism by which LetE augments transmission traits has not been elucidated (Bachman and Swanson 2004b). Homologs of letE have not been identified in other microbial genomes, and sequence analysis has provided little insight to its biochemical activity. Thus, additional research is necessary to determine how this protein functions and, furthermore, how LetE interacts with other regulators to coordinate L. pneumophila differentiation (Bachman and Swanson 2004b).

7.2. Carbon Storage Regulatory (Csr) System In a variety of microbes, the global regulatory system CsrA/CsrB functions posttranscriptionally to control stationary phase gene expression. Numerous cellular processes are regulated by this highly conserved system, including the inhibition of biofilm formation, gluconeogenesis, glycogen biosynthesis and catabolism, as well as the activation of glycolysis, acetate metabolism, flagellum biosynthesis, and motility (Suzuki et al. 2002). CsrA is a small effector protein that binds near the ribosomal binding site, and, depending on the target mRNA, either stabilizes

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the transcript or promotes transcript decay (Romeo 1998). CsrB is an untranslated RNA that contains 18 imperfect repeats located in the predicted loop regions of the RNA molecule (Romeo 1998). CsrB forms a globular complex with approximately 18 CsrA molecules, likely facilitated through binding the repeat elements (Romeo 1998). As a result, CsrB antagonizes the activity of CsrA by sequestering the molecule away from its target mRNAs, thus reducing the concentration of free CsrA (Romeo 1998). A homolog of the E. coli csrA gene was identified in L. pneumophila and shown to be essential for replication in broth culture, macrophages, and A. castellanii (Fettes et al. 2001; Molofsky and Swanson 2003; Forsbach-Birk et al. 2004). Genetic data indicate that, in L. pneumophila, every transmission trait induced by the LetA/LetS two-component system is repressed by CsrA (Fettes et al. 2001; Molofsky and Swanson 2003). In addition, the loss of CsrA activity bypasses all transmission trait defects displayed by a letA mutant, suggesting LetA functions to alleviate CsrA repression (Molofsky and Swanson 2003). The model predicts that when nutrients are depleted, RelA produces (p)ppGpp, thereby activating LetA/LetS. The two-component system then activates a hypothetical CsrB, which titrates CsrA away from its mRNA targets, allowing transmissive phase traits to be induced (Figure 6.2). Although specific mRNA targets of CsrA have not been identified, the CsrArepressed transmission traits of cytotoxicity, lysosomal evasion, and motility all depend on the efficient transcription of the flagellar sigma factor, FliA (Fettes et al. 2001; Hammer et al. 2002; Molofsky and Swanson 2003). Furthermore, over-expression of csrA leads to a reduction in fliA and flaA transcript levels (Fettes et al. 2001). By analogy to other prokaryotic CsrA/CsrB regulatory systems, it is predicted that L. pneumophila CsrA inhibits the stability or translation of fliA mRNA, thus affecting transcription of flaA and the expression of motility and transmission phenotypes (Romeo 1998). While data suggest that several copies of csrA are present in L. pnuemophila, the significance of multiple csrA homologs is unclear (Brassinga et al. 2003). Additional research will provide insight into how CsrA regulates its mRNA targets to enhance the fitness of L. pneumophila and control its differentiation.

7.3. Hfq and Small RNAs (sRNAs) In addition to the CsrA/CsrB global regulatory system, prokaryotes can employ other non-coding RNAs to modulate gene expression (Gottesman 2004; Majdalani et al. 2005). These sRNAs require the Hfq chaperone protein, and, through complementary base pairing with mRNAs, can modify either the translation or stability of their mRNA targets (Gottesman 2004; Majdalani et al. 2005). Data indicate that Hfq contributes to virulence in a number of bacterial pathogens, presumably by altering the interaction of sRNAs with rpoS, thus affecting rpoS translation (McNealy et al. 2005). Recently, a hfq homolog was identified in L. pneumophila, and the gene appears to be temporally regulated by both RpoS and LetA (McNealy et al. 2005).


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In the replicative phase, RpoS induces the expression of hfq (McNealy et al. 2005). Exponential phase transcripts, such as csrA and fur (ferric uptake regulator), are then stabilized by Hfq and most likely by sRNAs (McNealy et al. 2005). Upon entering stationary phase, (p)ppGpp accumulates and the LetA/LetS two-component system is activated (Hammer and Swanson 1999; Hammer et al. 2002). As a result, LetA induces transmissive phase traits, while directly or indirectly repressing hfq transcription (McNealy et al. 2005). Although sRNAs have not been found in L. pneumophila, they could be identified using Hfq as a tool, then their role in post-transcriptional regulation of differentiation could be subsequently examined.

8. Regulation of the Dot/Icm Type IV Secretion System To escape the endocytic pathway and establish a replicative vacuole, L. pneumophila depends on a group of 26 genes designated dot/icm (defect in organelle trafficking/intracellular multiplication) (Sexton and Vogel 2002). This family of genes has homology to bacterial conjugation systems and mediates the transfer of mobilizable plasmids between bacterial strains (Sexton and Vogel 2002). In addition, the type IV secretion system encoded by the dot/icm genes is predicted to translocate effector molecules into host cells (Sexton and Vogel 2002). At present, information regarding factors that regulate the timing and expression of genes required for assembling the secretion apparatus and its secreted effectors is limited. A series of nine icm::lacZ translational fusions were constructed, and examination of their temporal expression indicated that, in broth culture, several icm genes show a modest increase in their expression level upon entering stationary phase, including: icmF, icmM, icmP, icmR, and icmT (Gal-Mor et al. 2002). Subsequent studies demonstrated that LetA moderately regulates the expression of icmP, icmR, and icmT, while RelA and RpoS have only minor effects on dot/icm expression (Zusman et al. 2002; Gal-Mor and Segal 2003a). In contrast, letA mutants show a substantial decrease in dotA transcription, indicating that LetA may regulate a subset of dot/icm genes (Lynch et al. 2003). While each of the previously described studies have implicated indirect regulators of dot/icm genes, the only evidence for direct regulation is by the response regulator CpxR (Gal-Mor and Segal 2003b). CpxR and its cognate sensor kinase CpxA constitute a classic two-component system whose autophosphorylation is stimulated by stress signals (Gal-Mor and Segal 2003b). Data indicate that CpxR directly regulates icmR gene expression, and, while likely indirect, can also moderately induce the icmV-dotA and icmW-icmX operons. Nevertheless, CpxR is dispensable for intracellular growth in both human-derived macrophages and A. castellanii, suggesting that dot/icm genes must be positively regulated by other factors to enable L. pneumophila to establish a replicative vacuole (Gal-Mor and Segal 2003b).

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9. Genomic Methods of Studying Differentiation When confronted with environmental fluctuations, L. pneumophila must coordinately regulate its gene expression profile to enhance its fitness and adaptability. Since a large number of genes are dedicated to controlling differentiation, and a diverse repertoire of genes are expressed during the different phenotypic states, modern techniques have been applied to allow a comprehensive analysis of L. pneumophila biology. Recently, the complete genome sequences of L. pneumophila strains Lens, Paris, and Philadelphia 1 were published and many genes predicted to promote microbial adaptation were identified, including six sigma factors, 13 histidine kinases, 14 response regulators, and 23 members of the GGDEF-EAL family of regulators (Cazalet et al. 2004; Chien et al. 2004). A comparison of the genome sequences of Lens, Paris, and Philadelphia 1 revealed a remarkable plasticity and diversity of L. pneumophila, two characteristics thought to enhance the versatility of the microbe. In addition, genome-wide promoter trap strategies have been utilized to discern L. pneumophila genes that are specifically expressed during intracellular replication; this class of factors includes redox proteins, a response regulator and sensor kinase, several heavy metal transporters, and a gene homologous to the Pht family of transporters, smlA (reassigned phtK) (Rankin et al. 2002). Furthermore, proteomic analysis of L. pneumophila grown under different conditions has identified approximately 130 proteins, several of which are associated with virulence (Lebeau et al. 2005). In the future, research that is directed toward the global analysis of the L. pneumophila genome and proteome may identify regulatory factors that promote survival under disparate environmental conditions, as well as components essential to its pathogenesis.

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Nystrom T (2004) Growth versus maintenance: a trade-off dictated by RNA polymerase availability and sigma factor competition? Mol Microbiol 54:855–862 Oyston PCF, Sjostedt A, and Titball RW (2004) Tularaemia: bioterrorism defence renews interest in Francisella tularensis. Nat Rev Microbiol 2:967–978 Pao SS, Paulsen IT, and Saier MH, Jr. (1998) Major Facilitator Superfamily. Micro Mol Biol Rev 62:1–34 Perraud A, Weiss V, and Gross R (1999) Signalling pathways in two-component phosphorelay systems. Trends Microbiol 7:115–120 Rankin S, Li Z, and Isberg R (2002) Macrophage-induced genes of Legionella pneumophila: protection from reactive intermediates and solute imbalance during intracellular growth. Infect Immun 70:3637–3648 Ristroph JD, Hedlund KW, and Gowda S (1981) Chemically defined medium for Legionella pneumophila growth. J Clin Microbiol 13:115–119 Romeo T (1998) Global regulation by the small RNA-binding protein CsrA and the non-coding RNA molecule CsrB. Mol Microbiol 29:1321–1330 Rowbotham TJ (1986) Current views on the relationships between amoebae, Legionellae and man. Isr J Med Sci 22:678–689 Sauer JD, Bachman MA, and Swanson MS (2005) The phagosomal transporter A couples threonine acquisition to differentiation and replication of Legionella pneumophila in macrophages. Proc Natl Acad Sci USA 102:9924–9929 Sexton JA, and Vogel JP (2002) Type IVB secretion by intracellular pathogens. Traffic 3:178–185 Sturgill-Koszycki S, and Swanson MS (2000) Legionella pneumophila replication vacuoles mature into acidic, endocytic organelles. J Exp Med 192:1261–1272 Suzuki K, Wang X, Weilbacher T, Pernestig A-K, Melefors O, Georgellis D, et al. (2002) Regulatory circuitry of the CsrA/CsrB and BarA/Uvry systems of Escherichia coli. J Bacteriol 184:5130–5140 Tesh MJ, Morse SA, and Miller RD (1983) Intermediary metabolism in Legionella pneumophila: utilization of amino acids and other compounds as energy sources. J Bacteriol 154:1104–1109 Uhl MA and Miller JF (1996) Integration of multiple domains in a two-component sensor protein: the Bordetella pertussis BvgAS phosphorelay. EMBO J 15(5):1028–1036 Watarai M, Andrews HL, and Isberg RR (2001) Formation of a fibrous structure on the surface of Legionella pneumophila associated with exposure of DotH and DotO proteins after intracellular growth. Mol Microbiol 39:313–329 Wieland H, Faiglea M, Lang F, Northoffa H, and Neumeister B (2002) Regulation of the Legionella mip-promotor during infection of human monocytes. FEMS Microbiol Letters 212:127–132 Wieland H, Ullrich S, Lang F, and Neumeister B (2005) Intracellular multiplication of Legionella pneumophila depends on host cell amino acid transporter SLC1A5. Mol Microbiol 55:1528–1537 Wolfe AJ (2005) The acetate switch. Micro Mol Biol Rev 69:12–50 Zusman T, Gal-Mor O, and Segal G (2002) Characterization of a Legionella pneumophila relA insertion mutant and roles of RelA and RpoS in virulence gene expression. J Bacteriol 184:67–75.

7 Microbial Physiology Paul S. Hoffman

1. Introduction The genus Legionella is composed of over 48 named species and numerous serogroups of Gram-negative bacilli that are highly successful in colonizing natural aquatic environments worldwide. Members of this genus include species that exhibit an obligate requirement for a eukaryotic cell in which to replicate (do not grow in vitro), as well as species that can be grown in vitro on laboratory medium. All of these organisms exhibit a strictly respiratory form of metabolism (Hoffman and Pine, 1982; Hoffman, 1984) and utilize organic acids and amino acids as carbon and energy sources, which they obtain from their host. Much of what we know about these organisms is derived from studies of serogroup 1 L. pneumophila, the species most commonly associated with human disease. Furthermore, most of the basic information on bacterial physiology and metabolism has been gained from in vitro studies and little attention has been paid to what happens in vivo. Several recent developments in this field have caused us to re-examine previous in vitro findings, as we now know that L. pneumophila and several other species are dimorphic and cycle between vegetative replicating forms within host cells and transmissible, highly infectious cyst-like forms (Garduño et al., 2002; Greub and Raout, 2003). The developmental cycle is described in more detail in Chapter 4. In the case of intracellular replicating forms (RFs), they are indistinguishable from in vitro grown vegetative bacteria with respect to cell wall morphology (typical Gram-negative cell wall structure) and it has been generally assumed that their nutritional requirements are also similar to those of in vitro grown bacteria. In order to replicate and obtain nutrients from the infected host cell, the bacteria subvert the host cell by altering organelle trafficking to block fusion of secondary lysosomes with the phagosome and then remodel the endosome to permit bacterial multiplication. In natural protozoan hosts, the legionellae establish near symbiotic relationships with their hosts and even can persist in amoebic cysts. Late in intracellular infection when nutrients are exhausted, the bacteria differentiate into survival forms that when released from spent hosts are able to persist for extended periods in water or biofilms. These extracellular planktonic forms are morphologically distinct from vegetative 113

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bacteria and display thickened cell walls and cytoplasm occupied by inclusions of poly-␤-hydroxybutyrate (PHBA). The second major development in this field has been the completion and annotation of three complete genome sequences (Cazelet et al., 2004; Chien et al., 2004; see Chapter 1). The genome information has permitted a cataloging of genes and gene systems and in silico reconstruction of the metabolic pathways of this pathogen. This chapter will focus on basic features of physiology and metabolism within the context of a dimorphic lifestyle.

2. Intracellular Growth When intracellular, the legionellae acquire nutrients from the host cell that includes trace metals such as iron, essential amino acids, and amino acids that serve as key carbon and energy sources. In the case of mammalian hosts (lymphoid and non-lymphoid cells), which are grown in nutrient complete tissue culture medium, the intracellular bacteria rely on the host’s ability to transport nutrients from the tissue culture medium. In this regard, Wieland et al. (2005) recently demonstrated that monocyte transporter SLC1A5 (amino acid transporter) was required for intracellular growth of L. pneumophila. Presumably, L. pneumophila will not grow in starved hosts. While L. pneumophila can multiply in some host cells treated with cycloheximide to inhibit host protein synthesis (Garduño et al., 1998b), apparently protein synthesis is required in amoebic hosts (Fields, 1996). These differences likely reflect the differing nutrient sources between mammalian cells and freshwater amoebae, the latter associated with acquiring nutrients from phagolysosomes in which bacteria are digested as a source of nutrients, while for the former mammalian cell lines, transporters continue to take up amino acids as the cells are not immediately killed by protein synthesis inhibitors. Few studies have examined nutrient uptake by L. pneumophila. While the genome sequence indicates that L. pneumophila possesses ⬃12 classes of ABC transporters and amino acid permeases, the extent to which they contribute to solute uptake is not known. Based on comparative genome data, L. pneumophila contains ⬃16 major facilitator (MFP) genes and drug efflux systems and 9 of these are shared with related gamma proteobacterium C. burnetii (Cazalet et al., 2004). MFP transporters are often involved in sugar transport and drug efflux; however, the legionellae do not utilize sugars (Hoffman, 1984). Interestingly, of the 116 genes conserved among L. pneumophila, Coxiella burnetii (close relative), and Francisella tularensis, 28 encode transporters (Neurogadgets.com), which suggests a common strategy in acquiring nutrients from the host. The conserved set of transporters might be the result of lateral gene transfer or perhaps by convergent evolution of transporters that enable intracellular parasites to exploit specific amino acid pools common to eukaryotic cells. Studies by Sauer et al. (2005) identified a major facilitator transporter (PhtA) that was associated with threonine metabolism. Threonine is one of the required amino acids. Mutants in phtA required excess threonine or threonine-containing peptides for

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growth, suggesting that these conserved MFPs might be important in the uptake of amino acids. Intracellular bacteria also require iron, which they acquire from the host (iron metabolism is described in detail in Chapter 2. Early studies by Byrd and Horwitz (1989) showed that interferon gamma–treated macrophages restricted synthesis of transferrin receptors and limited iron uptake, and under these conditions growth of L. pneumophila was inhibited. Transporters for many other metals (Ni, Cu, Zn, etc.) for sodium and potassium efflux and for osmoprotection are all found with the genome of L. pneumophila.

3. Morphology, Ultrastructure, and Surface Proteins Legionella pneumophila displays a typical Gram-negative envelope in which the inner membrane and outer membrane can be readily distinguished in thin sections by electron microscopy (see Figure 7.1a). In some thin sections of L. pneumophila, outer membrane blebs are observed, a feature typical of most Gram-negative bacteria. In contrast, the cyst form contains a thick cell wall; laminations of intracytoplasmic membranes, and a cytoplasm occupied with inclusions of PHBA (see Figure 7.1b,c,d). While biochemical studies have been done on the composition of cell wall components from vegetative forms, little

A

B

Thin outer membrane and defined periplasm

Thick cell wall.

PHB

.

C

D

FIGURE 7.1. Morphological features of vegetative and cyst forms of L. pneumophila. Ultrathin sections of vegetative L. pneumophila (A) and cyst forms (B, C, and D). The vegetative bacteria have a thing outer membrane and defined inner membrane. The cytoplasm generally contains no inclusion bodies. In contrast, cyst forms have a thickened cell wall and laminations of intracytoplasmic membranes. The cytoplasm begins to accumulate inclusions that contain PHBA (poly-␤-hydroxybutyrate).

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information exists on the biochemical composition of the cyst forms (Faulkner and Garduño, 2002). The thickened cell wall and loss of typical Gram-negative cell wall architecture resembles cyst forms of other bacterial species such as Azotobacter and Rhotobacter. Developmental forms are discussed in more detail in Chapter 4.

3.1. Envelope Structure and Lipopolysaccharide (LPS) The outer membrane is composed of lipopolysaccharide which contains the serogroup-specific antigen that has been used for diagnosis of Legionnaires’ disease. The LPS is of low endotoxicity, relative to the LPS of Salmonella spp. (Neumeister et al., 1998). The O-antigen repeat from serogroup 1 strains of L. pneumophila (legionaminic acid) is 5-acetamidino-7-acetamido-8-O-acetyl3,5,7,9-tetradeoxy-L-glycero-D-galacto-nonulosonic acid, an N- and O-acylated derivative of legionaminic acid (Zou et al., 1999). The O-acetylase responsible for acetylation of LPS as determined from mutational analysis is located within an LPS biosynthesis locus (Luck et al., 2001). Studies with monoclonal antibodies reveal strain variations in LPS, but most human cases are caused by the Mab2 group which includes the Philadelphia-1 strain that caused the original outbreak of Legionnaires’ disease. While early studies suggested that variations in LPS composition contributed to virulence, more recent studies have suggested that loss of O-acetylation and changes in LPS composition did not affect infectivity as determined with natural amoebic hosts and cell lines (Luck et al., 2001). The LPS from L. pneumophila stimulates antibody production when injected into rabbits, the basis of serological testing in this genus. The low endotoxicity of the LPS might be attributable to low stimulation of toll like receptor 4 (TLR4) and more recent studies suggest that CD-14 and TLR2 are involved in signal transduction by LPS (Braedel-Ruoff et al., 2005).

3.2. Outer Membrane Proteins Outer membrane protein profile studies have shown that all serogroups of L. pneumophila express a dominant 28–29 kDa protein (OmpS) (Butler et al., 1985). Legionella micdadei also expresses a dominant 35 kDa protein (Amano and Williams, 1983). Other examined species of Legionella express outer membrane proteins, but no major dominant protein. It has been presumed that the unusual 28 kDa protein of L. pneumophila and perhaps the 35 kDa protein of L. micdadei contribute to virulence of these strains. Early studies by BellingerKawahara and Horwitz (1990) showed that OmpS bound complement factors that promoted attachment and invasion of host cells. This group also presented evidence to suggest that OmpS was a cationic selective porin (Gabay et al., 1985). Radiolabeling with 35S-cysteine revealed that OmpS was a cysteine-rich protein, a feature shared with another intracellular parasite Chlamydia trachomatis (Butler et al., 1985). Molecular and biochemical analysis of OmpS structure established that the protein was composed of three subunits bound by


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inter-chain disulfide bonds (Butler and Hoffman, 1990). One of the three subunits is anchored to the underlying peptidoglycan via a peptide bond between an N-terminal glycine of OmpS and diaminopimelic acid of the peptidoglycan (Butler and Hoffman, 1990; Hoffman et al., 1992). Each monomer of OmpS contains four cysteine residues which are predicted to participate in disulfide bonding. Studies by Weeratna et al. (1994) showed that purified OmpS was a major protective antigen of cellular immunity as demonstrated in guinea pigs, and by lymphocyte proliferative responses from humans up to seven years postinfection. The early observations regarding complement binding by OmpS could not be reproduced in a recent study that suggested that complement levels in the human lungs would preclude a major role in pathogenesis (Weissgerber et al., 2003). However, studies with HeLa cell attachment indicated that OmpS promoted attachment (independent of complement receptors or added complement), but not invasion and further that the role of OmpS in attachment varied among strains of L. pneumophila (Garduño et al., 1998b). There have been no studies of the outer membrane proteins from other species of Legionella, so it is not possible to predict how outer membrane proteins in other species function in attachment or pathogenesis. The MIP (macrophage infectivity potentiator) protein is also found in the outer membranes of all species of Legionella (Engleberg et al., 1989). The protein exhibits peptidyl-prolyl-cis/trans isomerase activity and may be involved in proper folding of outer membrane proteins or secreted proteins (Fischer et al., 1992). Other prominent proteins include the 19 kDa lipoprotein which may also be covalently bound to the underlying peptidoglycan and proteins associated with flagella motors, types I, II, and IV secretion systems, and the surface-associated chaperonin protein Hsp60 which promotes invasion (Garduño et al., 1998a), alters organelle trafficking in HeLa cells, and promotes production of cytokines including IL-1␤ by murine monocytes (Retzlaff et al., 1996).

3.3. Secreted Proteins Early work by Thorp and Miller (1981) showed that culture supernatants from L. pneumophila contained enzyme activities for extracellular protease, phosphatase, lipase, deoxyribonuclease, ribonuclease, and beta-lactamase. The extracellular protease is similar to elastase of Pseudomonas aeruginosa and exhibits hemolytic as well as proteolytic activity (Keen and Hoffman, 1989). Protease mutants are attenuated for virulence in a guinea pig model of infection (Moffat et al., 1994). The protease is secreted into the host cell phagosome during infection, but how this protein functions in pathogenesis has not been fully explored. The protease is secreted by the type II secretion system of L. pneumophila as are many of the extracellular enzymes, including acid phosphatase, and several phospholipases (Aragon et al., 2001; Flieger et al., 2001). Mutants deficient in acid phosphatase activity were fully virulent in macrophage cell lines and amoeba. With respect to lipases, L. pneumophila produces several classes of these enzymes that can be both cell associated and secreted (Flieger et al., 2001). One of the

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phospholipases (phospholipase C) is secreted by the TAT (twin arginine secretion system) (Rossier and Cianciotto, 2005). Additionally, L. pneumophila secretes phospholipase A and several lysopholipases and together with cell-associated lipases might play important roles in fatty acid metabolism by intracellular bacteria. The role of the many phospholipases during intracellular growth has not been investigated.

3.4. Motility and Chemotaxis Several studies have shown that L. pneumophila is motile when grown at low temperature (⬍30° C) and non-motile during exponential phase at 37° C (Dietrich et al., 2001). However, as bacteria enter post-exponential growth, they become motile. This is unusual as most bacteria are motile during exponential phase of growth. During intracellular growth, bacteria become motile late in infection (37° C) and motility is readily apparent within phagosomes of infected host cells (cover slip assays of infected monolayers). When bacteria are ultimately released from spent host cells, motility is maintained for ca 24 h. It has been speculated that released bacteria require motility in order to seek out new host cells or to maneuver around in biofilms. However, a search of the genome sequence of L. pneumophila reveals that L. pneumophila lacks all of the chemotaxis genes (methyl-accepting chemoreceptors, CheA, CheW, CheY, CheB, CheR, CheZ) and others such has hybrid CheV proteins required for chemotaxis. Additionally, L. pneumophila is not chemotactic by soft agar assays that are typically used to demonstrate chemotaxis by other bacteria. Therefore, in the absence of chemotaxis, bacterial motility is random and probably not used to seek out new hosts or sources of nutrients in the extracellular milieu. It is possible that motility during late stages of intracellular infection prevents the bacteria from becoming adherent to the phagosome membrane or potentially that the swimming mass of bacteria communicate, perhaps through some form of quorum sensing that may synchronize differentiation to cyst-like planktonic survival forms. Non-motile mutants are unaltered in intracellular growth, though several studies suggest that flagella might improve the odds of finding a host cell under conditions where population densities of suitable hosts are high (Dietrich et al., 2001). At 37° C, post-exponential expression is under the control of the stationary phase sigma factor RpoS, the nitrogen regulator RpoN, and the sigma-28 alternative sigma factor (Molofsky and Swanson, 2004). Moreover, flaA-gfp (flagellin subunit fused with green fluorescent protein reporter) fusions have been used to track post-exponential expression of flagella and to elucidate the regulatory factors. The regulation of post-exponential gene expression is detailed in chapter ([Swanson]). In addition to flagella, L. pneumophila also expresses type IV pili that may also play a role in attachment to host cells or possibly persistence in biofilms (Stone and Abu Kwaik, 1998). It is not known, however, if pili are present on the surface of planktonic cysts.


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4. Respiratory Metabolism Members of the genus Legionella exhibit a strictly respiratory form of metabolism and do not grow anaerobically. Moreover, anaerobic respiration with nitrate or other electron acceptors has not been demonstrated. The electron transport chain is composed of cytochromes of the b, c, aa3, and d types and the legionellae are cytochrome oxidase positive. The electron transport chain is branched with cytochrome c⬘/aa3 (cytochrome c oxidase complex) and cytochrome d as terminal electron acceptors (Hoffman and Pine, 1982). Each of these terminal oxidases binds carbon monoxide as validated by laser-assisted photochemical action spectral studies (author’s unpublished studies). It is likely that each of the terminal oxidases exhibits a different Km for oxygen and functions under different oxygen tensions or stages of growth. Few studies have examined the bioenergetics of L. pneumophila either in vivo or in vitro. It is likely that late in intracellular growth molecular oxygen becomes limiting and leads to changes in the types and levels of expression of the terminal oxidases. The major substrates respired by membrane vesicles include NADH, succinate, malate, and ascorbate TMPD (N,N, N⬘,N⬘-tetramethyl-p-phenylenediamine). Biogenesis studies of cytochrome c show that proper function of the electron transport chain is important for infectivity of L. pneumophila (Naylor and Cianciotto, 2004). While no studies have examined the energy conservation efficiency of the L. pneumophila electron transport chain, it is likely that protons are pumped via the NADPH dehydrogenase complex, the quinone cytochrome b complex, and possibly a single proton is pumped from the cytochrome oxidase complex. Further studies will be required to assess to what extent a branched respiratory chain influences respiratory activity and energy conservation efficiency.

5. Central Intermediary Metabolism From whole genome analysis and from early biochemical and radiotracer studies we know that L. pneumophila contains a complete glycolytic pathway as well as key components of the pentose phosphate pathway and Entner Doudoroff pathway (see Figure 7.2). L. pneumophila lacks sugar transporters and must rely on gluconeogenesis for synthesis of sugars for biosynthesis of peptidoglycan, LPS, ribose, and deoxyribose and other cellular components. The bacteria can be grown in chemically defined medium and the addition of glucose to this medium does not change the growth rate (Ristrof et al., 1981; Warren and Miller, 1979). However, no studies have examined the nutritional requirements of L. pneumophila during intracellular growth or whether phosphorylated or nucleotide sugars might be utilized under these conditions. Generally bacteria that must synthesize sugars from Krebs cycle intermediates express several major gluconeogenic enzymes. There are several major enzymes of gluconeogenesis that connects the Krebs cycle with the gluconeogenic pathway. PEP carboxylase, which converts oxaloacetate (OAA) to phosphoenol pyruvate (PEP), has been

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FIGURE 7.2. Metabolic pathways of L. pneumophila. The metabolic pathways depicted include the Krebs cycle, Entner Doudoroff (ED), Pentose Phosphate and Embden Myerhof pathways. The enzyme abbreviations used are common and key metabolic reactions are indicated by thickened arrows. Key enzymes of these pathways are discussed in the text. Legionella lacks transporters for sugars and therefore must rely on gluconeogenesis for biosynthesis of sugars.

measured in cell free extracts (specific activity for this enzyme is 8 nmoles per min per mg of protein), and a second enzyme of this pathway, PEP carboxy kinase, was 8.4 nmoles per min per mg protein. Another major enzyme is PEP synthase which produces PEP by ATP-dependent phosphorylation of pyruvate. Pyruvate carboxylase produces OAA from pyruvate with ATP and CO2 and exhibits a high specific activity (140 nmoles per min per mg protein). The abundance of these classes of enzymes strongly supports a gluconeogenic metabolism


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and suggests that much of the carbon entering the Krebs cycle is directed to synthesis of glucose, fructose, and ribose sugars. L. pneumophila contains a complete catabolic Embden Myerhof pathway, but the direction of the pathway (catabolic versus gluconeogenic) is determined by the specific activities of key enzymes at several points in this pathway. In this regard, the key metabolic enzyme of the gluconeogenic pathway is fructose 1,6 biphosphatase whose activity in cell extracts from L. pneumophila was tenfold higher than that measured for phosphofructokinase, the key catabolic enzyme that forms fructose 1,6 biphosphate. Interestingly, the fructose 1,6 biphosphatase gene is not present in the genomes of all three sequenced strains of L. pneumophila and in close relative Coxiella burnetii. The lack of an annotated gene for an enzyme activity is not uncommon as in silico pathway reconstructions have demonstrated for other bacteria. There are generally three possible explanations assuming the enzyme data are robust: (i) the functional gene might have diverged sufficiently that it is not picked up on BLAST searches; (ii) a paralog enzyme of related function might catalyze the reaction; or (iii) an enzyme that is unidirectional in some bacteria may be bidirectional or bifunctional in other bacteria. In this regard, a further examination of the 6-phosphofructokinase enzyme of L. pneumophila and C. burnetii, which typically catalyzes the forward reaction, indicates that the enzyme is of the PfkA class that uses PPi (pyrophosphate) instead of ATP. These enzymes are bifunctional (catalyzing the phosphorylation as well as the phosphatase activity). In contrast, enzymes of the PfkB class are not reversible and in enteric bacteria the phosphofructokinase enzyme catalyzes the formation of fructose 1,6 biphosphate and the fructose 1,6-biphosphatase catalyzes formation of F6P. Since this enzyme step is critical in synthesis of fructose-6-P (see Figure. 7.2), it is likely that essentiality testing would demonstrate the necessity of pfkA in gluconeogenesis. Further studies with purified PfkA would be required to formally test substrate specificities and enzyme direction. A second key gene that is absent in L. pneumophila is 6-phosphogluconate dehydrogenase which decarboxylates 6-phosphogluconate to produce ribulose 5 phosphate, which is subsequently converted to ribose-5-P and used for synthesis of ribose and deoxyribose (DNA and RNA synthesis). In other bacteria, such as Helicobacter pylori, ribose sugars are produced from the products of the Entner Doudoroff pathway together with fructose 6-P that is generated via gluconeogenesis (Hoffman, 2001; Chalker et al., 2001). In H. pylori, fructose-1,6-biphosphatase activity is essential as mutations in the gene render H. pylori non-viable (Chalker et al., 2001). In this example, H. pylori lacks phosphofructokinase activity and is thereby blocked in the forward direction of glycolysis, despite its ability to utilize glucose. The major enzymes associated with the Pentose Phosphate Pathway of L. pneumophila (transketolase and transaldolase) catalyze many of the interconversions between fructose-6-P, glyceraldehyde-3-P (generated by the ED pathway) to produce all the intermediates required for synthesis of ribose and deoxyribose sugars, as well as 4-carbon compounds required for synthesis of vitamins and aromatic amino acids. Little attention has been paid to metabolic systems in L. pneumophila and metabolism is quite relevant to pathogenesis and persistence.

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This is especially true for the planktonic cyst form that exhibits no respiratory activity and is near dormant metabolically (Garduño et al., 2002). The Krebs cycle is complete and begins with the oxidative decarboxylation of pyruvate to produce acetyl CoA which condenses with oxaloacetate to form citrate (citrate synthase). However, the measured activity of pyruvate dehydrogenase was low (1.64 nmoles per min per mg protein) in cellular extracts suggesting that little pyruvate is converted to acetyl CoA (Keen and Hoffman, 1984). Generally pyruvate oxidation is required for generation of acetyl CoA to drive the Krebs cycle. However, it is quite likely that the required acetyl CoA is generated from fatty acid catabolism in concert with the many phospholipases noted for this organism. The deamination of L-aspartate, a major carbon source, produces sufficient oxaloacetate to condense with acetyl CoA to produce citrate. Such reactions would spare consumption of pyruvate by the Krebs cycle. Other organic acid intermediates of the Krebs cycle are derived from glutamate, serine, and related amino acids. In natural amoebic hosts, all of the amino acids would be obtained from the host. As mentioned earlier, much of the carbon required for biosynthesis of peptidoglycan, LPS, nucleic acids, and some of the amino acids associated with protein synthesis must be directed into the gluconeogenic route and this is supported by the high specific activities of gluconeogenic enzymes and Krebs cycle enzymes relative to those activities measured for the Embden Myerhof pathway. The complete Krebs cycle is depicted in Figure. 7.2. The aconitase enzyme that converts citrate to isocitrate has received some attention as it requires iron for biological activity. In iron-starved bacteria, the activity of this enzyme decreases and is part of the phenotype associated with iron-restricted growth. In addition to the oxoglutarate dehydrogenase system (dihydrolipoamide dehydrogenase and dihydrolipoamide succinyltransferase), L. pneumophila also contains the KorA and KorB subunits of oxoglutarate ferredoxin oxidoreductase that is commonly found in anaerobic bacteria. While the genes are present, direct enzymatic activity for these proteins has not been examined. It is possible that this alternate pathway functions under oxygen-limiting conditions that might exist during cyst morphogenesis late in the growth cycle in vivo. The POR and KOR enzymes are usually coupled with ferredoxin or flavodoxin that can function under anaerobic conditions. The specific activity for ␣-ketoglutarate dehydrogenase (NAD) was robust (⬃20 nmoles per min per mg protein) indicating that this is the major enzyme complex in bacteria grown in vitro. Most of the Krebs cycle enzymes are of high specific activity relative to glycolytic enzymes. Neither the genome sequence nor direct enzyme assay found any evidence for the glyoxylate bypass in L. pneumophila (Keen and Hoffman, 1984; Cazalet et al., 2004). While the isocitrate dehydrogenase exhibited specificity for NADP, the malate dehydrogenase exhibited specificity for NAD. One of the most active enzymes detected in cell free extracts of L. pneumophila was glutamate-aspartate transaminase which transfers the amino group from glutamate to oxaloacetate to produce aspartate. In order for there to be available oxaloacetate for this reaction (assuming an abundance of glutamate), conversion of serine to pyruvate and subsequent CO2 fixation to


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oxaloacetate would be required. It is not clear how the carbon and nitrogen balance is maintained or when in the growth cycle synthesis of storage polymers of poly-␤-hydroxybutyrate is initiated. Generally synthesis of PHBA occurs under conditions of high carbon and low nitrogen.

6. Biosynthetic Capacity and Amino Acid Auxotrophies Most species of Legionella can be grown in vitro on specialized yeast extract–based medium supplemented with ACES buffer, ␣-ketoglutarate, L-cysteine, and ferric pyrophosphate (BCYE␣ medium) (Feeley et al., 1979). This medium has not changed in 25 years. Early studies showed that charcoal and ␣ketoglutarate function by quenching free radical reactions and thereby detoxified the medium of toxic reduced forms of oxygen (Hoffman et al., 1983). Several studies have further dissected the nutritional requirements for L. pneumophila and chemically defined media have been developed (Warren and Miller, 1979; Ristroff et al., 1981). Radiotracer studies confirmed an inability by L. pneumophila to transport glucose (Keen and Hoffman, 1984). These studies show that most strains require the amino acids threonine, cysteine, methionine, leucine, isoleucine, valine, arginine, and serine. Additionally, proline, glutamate, and phenylalanine or tyrosine stimulate growth. While not required, guanine has been shown to stimulate growth of some strains of L. pneumophila and most other species of Legionella. The legionellae do not display requirements for purines, pyrimidines, or vitamins, and from in silico metabolic pathway reconstructions appear to possess the requisite biosynthetic pathways. With the exception of L. oakrdgensis and L. spiritensis, all species exhibit an obligate requirement for the amino acid cysteine, a distinguishing feature for members of this genus. Analysis of the three sequenced L. pneumophila genomes indicates that the genes encoding cysteine biosynthetic enzymes are absent and support early biochemical tests that reached a similar conclusion (Hoffman, 1984). The apparent inability of L. pneumophila to transport the oxidized form of cysteine (L-cystine), which is most abundant under aerobic conditions, likely restricts the bacteria to an intracellular existence and may well be one of several amino acids likely to transmit the germination signal to the dormant cysts ingested by amoebae or human macrophages. Analysis of the annotated L. pneumophila genomes reveal some gaps in biosynthetic pathways suggesting that new genes and enzymes catalyzing these reactions will ultimately be discovered (KEGG.com). In silico pathway reconstructions are largely supported by early nutritional work and development of various chemically defined media. Very little is known about regulatory mechanisms and control of biosynthetic activities when L. pneumophila is intracellular. L. pneumophila is auxotrophic for the branch chain amino acids and the thiol amino acids methionine and cysteine. From the annotated L. pneumophila genomes, it is likely that the bacteria are also auxotrophic for phenylalanine and tyrosine as key enzymes from chorismate to phenylpyruvate appear to be absent. In the case of threonine auxotrophy, theonine synthetase is present as well as

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threonine dehydrogenase; however, biosynthetic enzymes forming the substrates for these reactions appear to be absent in the genome sequence. Leucine dehydrogenase is the only enzyme of the isoleucine, leucine, and valine biosynthetic pathways, and genes required for early steps in these biosynthetic pathways are absent. The enzymes associated with biosynthesis of arginine and methionine are also absent. The only enzyme of the methionine biosynthetic pathway (cystathionine beta-lyase) which interconverts cystathionine and L-homocysteine appears to have few substrates. The enzymes for S-adenosyl methionine metabolism are present. The key enzymes of cysteine biosynthesis (serine transacetylase and o-acetylserine sulfhydrolase) are present in L. oakridgenesis but these activities are undetectable in L. pneumophila. The genes encoding these enzymes are also absent in the genome sequence. The requirement for excess L-cysteine in laboratory media is due to the spontaneous oxidation of L-cysteine to L-cystine (unavailable to the bacteria) and the establishment of an equilibrium between these two forms that remains steady at ⬃0.5 mM L-cysteine, sufficient to support growth of the bacteria. The amino acid auxotrophies might play an important role in preventing cysts from germinating outside of suitable host cells. Since cysts require the intracellular milieu in order to germinate into vegetative bacteria, detection of amino acids, particularly those that are essential, might be essential for germination and intracellular replication (Sauer et al., 2005). Similarly, an absence of these amino acids late in infection of host cells might provide signals required for cyst morphogenesis.

7. Protection from Oxidative Stress Bacteria living under aerobic conditions must deal with toxic effects of oxygen that can be generated exogenously or endogenously from oxidation reactions within the bacteria cell. Early studies established that L. pneumophila grew poorly on most laboratory medium and that the addition of starch or activated charcoal dramatically improved the ability of media to support good growth. L. pneumophila grows poorly on BCYE␣ medium when the charcoal is omitted. The charcoal was subsequently shown to quench free radical propagating reactions that produced reduced forms of oxygen (hydrogen peroxide, superoxide anions, singlet oxygen, and the highly reactive hydroxyl radical) (Hoffman et al., 1983). Therefore, L. pneumophila may lack a strong defense against exogenous toxic oxygen intermediates. In this regard, Pine et al. (1984, 1986) first described variations among Legionella species in expression of catalase and peroxidase enzymes. L. pneumophila expresses two catalase-peroxidase enzymes, KatA being localized to the periplasm and KatB located in the cytoplasm (Bandyopadhyay and Steinman, 2000). These proteins are 60% conserved in amino acid sequence, suggesting that they arose from a common gene. The levels of both enzymes increase post-exponentially and are regulated by stationary phase sigma factor RpoS which binds to stationary phase gene promoter sequences (a 10 hexamer—TATACT). These enzymes display strong peroxidase activity and


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weak hydroperoxidase activity (degrade hydrogen peroxide). Other species of Legionella are presumed to express a true catalase (strong activity with hydrogen peroxide). In addition to two catalase/peroxidase genes in L. pneumophila, these bacteria also express two superoxide dismutases, one CuZn located in the periplasm and the other FeSOD located in the cytoplasm (St. John and Steinman, 1996). Since superoxide anions do not readily pass through the cytoplasmic membrane, it is assumed that the CuZn enzyme is responsible for protection from exogenous superoxide anions while the Fe enzyme protects against endogenous superoxide radicals. Indeed, in other bacteria, redox active dyes that generate endogenous superoxide radicals have been used to delineate function for the cytoplasmic enzyme. The periplasmic CuZn enzyme also contains a promoter sequence recognized by RpoS and is expressed post-exponentially. The typical SoxRS regulator of superoxide dismutase gene expression under conditions of oxidative stress is absent in L. pneumophila. How oxidative stress signals drive expression of these genes remains to be determined. In general, stationary phase bacteria are more susceptible to oxidative stress which might in part be explained by lower respiration rate due to limiting oxidizable substrates. Under these conditions, oxygen diffusing into the bacterial cells now reacts with redox active components that would otherwise be protected by a more active respiratory system. Additionally, L. pneumophila expresses two alkylhydroperoxide reductase enzymes (AhpC), both located in the cytoplasm that may be even more important in protection from hydrogen peroxide and organic peroxides. Organic peroxides result from the reactions of toxic forms of oxygen with membrane components leading to formation of lipid peroxides. In the absence of protective enzymes, lipid peroxidation reactions will ultimately render bacterial membranes non-functional. AhpC enzymes are found in all organisms and exist as oligomers of ten subunits. These enzymes usually have a very high affinity for hydrogen peroxide (low Km), and in E. coli they have been shown to be more important than catalase in protection from hydrogen peroxide toxicity. This is because the Km for peroxide for catalase is in the mM range whereas that for AhpC is in the ␮M range. The AhpC1 enzyme of L. pneumophila has been shown to be up-regulated during intracellular infection of macrophages and may be important in protection from the respiratory bust (Rankin et al., 2002). Phylogenetic analysis of the AhpC1 class of peroxyredoxins suggests that thioredoxin and thioredoxin reductase make the enzyme active in an NADPHdependent reaction as demonstrated for H. pylori (Baker et al., 2001). The second AhpC (AhpC2) is similar to the complex AhpCAhpD found in Mycobacterium tuberculosis (Bryk et al., 2002). This enzyme is activated by components of the pyruvate or ␣-ketoglutarate dehydrogenase systems. Studies in the author’s lab suggest that at least one functional enzyme is required for viability. The regulation of all of the oxidative stress genes is not understood and may involve many regulatory factors from OxyR (redox sensitive regulator of catalase and AhpC in other bacteria), RpoS, and perhaps other regulatory factors such as the extracytoplasmic sigma factors (ECF factors).

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8. Heat Shock Response One of the earliest stress responses studied in detail in L. pneumophila was the heat shock response which is common to all living organisms. Interest in heat shock proteins (HSPs) was initially driven by the finding of L. pneumophila in hot water systems, hot water tanks, and industrial boilers. In E. coli, the heat shock response is controlled by the heat shock sigma factor RpoH or ␴32. Following heat shock, RpoH is activated and binds to promoter regions of genes containing heat shock promoter sequences, initiating the synthesis of some 20 or more proteins that have been named HSP. These include Hsp60 (GroEL) and Hsp70 (DnaK) which provide key chaperone functions (also called chaperonins) by binding to denatured proteins and facilitating their renaturation or decomposition. Under non-stressful growth conditions, GroEL and DnaK as well as most of the other HSPs are produced in very low, almost non-detectible levels in E. coli. In most bacteria, the expression of heat shock proteins is both dramatic and transient, and within 20 min of heat shock, the protein levels return to low baseline levels. In contrast, many HSPs of L. pneumophila are very abundant under all growth conditions and following heat shock only increase in level by ⬃2-fold. Genes encoding HSPs of L. pneumophila contain canonical heat shock promoter sequences and the rpoH gene is present in the chromosome (Hoffman et al., 1990). Attempts to knock out this gene have been unsuccessful and suggest that RpoH function must be required to maintain high expression levels of Hsp60 and DnaK. One of the unique features of the Hsp60 of L. pneumophila is its cellular location; whereas in E. coli, GroEL is exclusively located in the bacterial cytoplasm, the Hsp60 of L. pneumophila is located in the periplasmic space and on the bacterial surface (Figure 7.3) (Garduño et al., 1998a). Studies have shown that the L. pneumophila Hsp60 remains in the cytoplasm of E. coli when the htpAB locus is expressed in E. coli. In contrast, when groELS is expressed in L. pneumophila, the E. coli protein localizes to the periplasm and outer surface (unpublished observations from the author’s lab). These studies suggest that L. pneumophila must express a novel secretion system that is absent in E. coli that facilitates secretion of Hsp60 proteins into the periplasm. The surface location of Hsp60 can be demonstrated by treating whole cells of L. pneumophila with trypsin which degrades surface-located proteins. Bacteria so treated lose infectivity for HeLa cells suggesting that Hsp60 may be important in pathogenesis. Several lines of study have established that surface-located Hsp60 promotes invasion of HeLa cells by L. pneumophila and that Hsp60 affixed to latex beads are rapidly taken up by non-phagocytic HeLa cells (general test for invasions). Avirulent mutants, and in particular, Dot/Icm Type IVB secretion mutants, secrete Hsp60 into the periplasm, but not onto the bacterial surface (trypsin assay). Thus, a functional type IV secretion system is necessary for surface location of Hsp60 (see Figure 7.3). Further study is required to determine how Hsp60 interfaces with the Dot/Icm system and whether the secretion is direct or indirect.


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FIGURE 7.3. Surface location of Hsp60 in virulent (wild type) and avirulent (dotA mutant). Ultrathin sections were treated with anti-Hsp60 monospecific serum and developed with 10 nm gold-conjugated anti-rabbit globulin. The gold particles represent the positions of the Hsp60 molecules. The arrows point to the outer membrane (OM) and the inner membrane (IM). Note that during fixation, the inner membrane often contracts from the outer membrane and aids clarification of Hsp60 localization.

Hsp60 in virulent (wild type) and avirulent (dotA mutant).

9. Stationary Phase Genes and Cell Wall Assembly The transition of exponentially growing bacteria into stationary phase is coordinate with production of the stringent response regulatory ppGpp and ppGppp and the activation of stationary phase sigma factor RpoS (Molofsky and Swanson, 2004). In vivo, these activities together with two-component regulatory system LetA/S and integration host factor (IHF) coordinate the expression of many genes whose products are associated with the transition of vegetative bacteria into resilient cysts. This transition to cysts does not occur in vitro, suggesting that host cell signals or perhaps different nutritional conditions are required to deliver the developmental signal. There are several markers of the post-exponential transition that include FlaA (flagellin subunit) and MagA (gene of unknown function that is found in a 45 kb genome island common to some strains of L. pneumophila). Still other markers of differentiation include synthesis of inclusions of PHBA. There are four genes encoding PHBA synthases and all but one contains promoter sequences resembling RpoS-binding sites or IHF-binding domains. Similarly, the magA gene contains both IHF- and RpoS-binding sites and the levels of MagA are diminished in rpoS and ihfAB mutants (Hiltz et al., 2004). The extent to which these and other regulatory genes control stages of cyst morphogenesis are currently under study.

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10. Conclusions Legionella pneumophila and related species are essentially intracellular parasites of aquatic protozoa and have adapted a metabolic strategy that exploits the host cell. Generally the host must provide amino acids, peptides, fatty acids, organic acids, and trace metals to the intracellular bacteria. From these building blocks, the legionellae can synthesize all cellular components. From whole genome analyses, several unique adaptations have been discovered and more will likely arise from the active study of additional genome sequences that are in progress. The dimorphic growth cycle of L. pneumophila has also added to our knowledge of regulatory systems and survival mechanisms. The knowledge gained from studies of the cysts will likely lead to development of better biocides that might reduce bacterial numbers in environments close to humans and reduce the transmission of Legionnaire’s disease.

References Amano K, Williams JC 1983. Partial characterization of peptidoglycan-associated proteins of Legionella pneumophila. J. Biochem (Tokyo). 94:601–606. Aragon V, Kurtz S, Cianciotto NP (2001). Legionella pneumophila major acid phosphatase and its role in intracellular infection. Infect. Immun. 69:177–185. Baker LM, Raudonikinene A, Hoffman PS, and Poole LB (2001) An essential thioredoxindependent peroxiredoxin from Helicobacter pylori: genetic and kinetic characterization. J. Bacteriol. 183:1961–1973. Bandyopadhyay P, Steinman HM (2000) Catalase-peroxidases of Legionella pneumophila: cloning of the katA gene and studies of KatA function. J. Bacteriol. 182:6679–6686. Bellinger-Kawahara C, Horwitz MA (1990). Complement component C3 fixes selectively to the major outer membrane protein (MOMP) of Legionella pneumophila and mediates phagocytosis of liposome-MOMP complexes by human monocytes. J. Exp. Med. 172: 1201–1210. Braedel-Ruoff S, Faigle M, Hilf N, Neumeister B, Schild H (2005) Legionella pneumophila mediated activation of dendritic cells involves CD14 and TLR2. J Endotoxin Res. 11:89–96. Bryk R, Lima CD, Erdjument-Bromage H, Tempst P, Nathan C (2002) Metabolic enzymes of mycobacteria linked to antioxidant defense by a thioredoxin-like protein. Science. 295:1073–1077. Butler CA, Hoffman PS (1990) Characterization of a major 31 kilodalton peptidoglycanbound protein of Legionella pneumophila. J. Bacteriol. 172:2401–2407. Butler CA, Street ED, Hatch TP, Hoffman PS (1985) Disulfide-bonded outer membrane proteins in the genus Legionella. Infect. Immun. 48:14–18. Byrd TF, Horwitz MA (1989) Interferon gamma-activated human monocytes downregulate transferrin receptors and inhibit the intracellular multiplication of Legionella pneumophila by limiting the availability of iron. J. Clin Invest. 83:1457–1465. Cazalet C, Rusniok C, Bruggemann H, Zidane N, Magnier A, Ma L, Tichit M, Jarraud S, Bouchier C, Vandenesch F, Kunst F, Etienne J, Glaser P, Buchrieser C (2004) Evidence in the Legionella pneumophila genome for exploitation of host cell functions and high genome plasticity. Nat Genet. 36:1165–1173.


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Chalker AF, Minehart HW, Hughes NJ, Koretke KK, Brown JR, Lonetto MA, Warren PV, Stanhope MJ, Lupas A, Hoffman PS (2001) Systematic identification of unique essential genes in Helicobacter pylori by genome prioritization and allelic replacement mutagenesis J. Bacteriol 183:1259–1268. Chien M, Morozova I, Shi S, Sheng H, Chen J, Gomez SM, Asamani G, Hill GK, Nuara J, Feder M, Rineer J, Greenberg JJ, Steshenko V, Park SH, Zhao B, Teplitskaya E, Edwards JR, Pampou S, Georghiou A, I. C. Chou IC, Iannuccilli W, Ulz ME, Kim DH, GeringerSameth A, Goldsberry C, Morozov P, Fischer SG, Segal G, Qu X, Rzhetsky A, Zhang P, Cayanis E, De Jong PJ, Ju J, Kalachikov S, Shuman HA, Russo JJ (2004) The genomic sequence of the accidental pathogen Legionella pneumophila. Science. 305:1966–1968. Dietrich C, Heuner K, Brand B, Hacker J, and Steinert M (2001) The flagellum of Legionella pneumophila positively affects the early phase of infection of eukaryotic host cells. Infect. Immun. 69:2116–2122. Engleberg NC, Carter C, Weber DR, Cianciotto NP, Eisenstein BI (1989) DNA sequence of mip, a Legionella pneumophila gene associated with macrophage infectivity. Infect Immun. 57:1263–1270. Faulkner G, Garduño RA (2002) Ultrastructural analysis of differentiation in Legionella pneumophila. J Bacteriol 184: 7025–7041. Feeley JC, Gibson RJ, Gorman GW, Langford NC, Rasheed JK, Mackel DC, Baine WB (1979). Charcoal-yeast extract agar: primary isolation medium for Legionella pneumophila. J. Clin Microbiol. 10:437–441. Fields BS (1996) Molecular ecology of legionellae. Trends in Microbiol. 4:286–290. Fischer G, Bang H, Ludwig B, Mann K, Hacker J (1992) Mip protein of Legionella pneumophila exhibits peptidyl-prolyl-cis/trans isomerase (PPlase) activity. Mol Microbiol. 6:1375–1383. Flieger A, Gong S, Faigle M, Stevanovic S, Cianciotto NP, and Neumeister B (2001) Novel lysophospholipase A secreted by Legionella pneumophila. J. Bacteriol. 183:2121–2124. Gabay JE, Blake M, Niles WD, Horwitz MA (1985) Purification of Legionella pneumophila major outer membrane protein and demonstration that it is a porin. J. Bacteriol. 162:85–91. Garduño RA, Garduño E, Hoffman PS (1998a) Role of the Hsp60 chaperonin of Legionella pneumophila in mediating invasion in a HeLa cell model. Infect. Immun. 66:4602–4610. Garduño R, Quinn FD, Hoffman PS (1998b) HeLa cells as a model to study the invasiveness and biology of Legionella pneumophila. Can. J. Microbiology. 44:430–440. Garduño, RA, Garduño E, Hiltz M, and Hoffman PS (2002) Intracellular growth of Legionella pneumophila gives rise to differentiated form dissimilar to stationary phase forms. Infect. Immun. 70: 6273–6283. Greub G, Raoult D (2003) Morphology of Legionella pneumophila according to their location within Hartmanella vermiformis. Res Microbiol 154: 619–621. Hiltz MF, Sisson GR, Brassinga AKC, Garduño E, Garduño RA, Hoffman PS (2004) Expression of magA in Legionella pneumophila Philadelphia-1 is developmentally regulated and a marker of MIF formation. J. Bacteriol. 186:3038–3045. Hoffman, PS (1984) Bacterial Physiology. In C. Thornsberry et al. (eds.). Legionella, Proceedings of the 2nd International Symposium, ASM, Washington, DC, pp. 61–67. Hoffman PS (2001) Bacterial Physiology of Helicobacter pylori. In H. Friedman, P.S. Hoffman and Y. Yamamoto (eds.). Immunology and Pathogenesis of Helicobacter pylori. Kluwer Press, USA. Hoffman PS, and Pine L (1982) Respiratory physiology and cytochrome content of Legionella pneumophila. Curr. Microbiol. 7:351–356.

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Hoffman PS, Pine L, and Bell S (1983) Production of superoxide and hydrogen peroxide in medium used for culture of Legionella pneumophila: catalytic decomposition by charcoal. Appl. Environ. Microbiol. 45:784–791. Hoffman PS, Houston L, Butler CA (1990) Legionella pneumophila htpAB heat shock operon: nucleotide sequence and expression of the 60-kilodalton antigen in L. pneumophila-infected HeLa cells. Infect. Immun. 58:3380–3387. Hoffman PS, Seyer JH, and Butler CA (1992) Molecular characterization of the 28 and 31 kilodalton subunits of the Legionella pneumophila porin. J. Bacteriol. 174:908–913. Keen MG, Hoffman PS (1984) Metabolic pathways and nitrogen metabolism in Legionella pneumophila. Curr. Microbiol. 11:81–88. Keen MG, Hoffman PS (1989) Characterization of a protease mutant of Legionella pneumophila and demonstration that the protease is responsible for the hemolytic and cytotoxic phenotyes. Infect. & Immun. 57:732–738. Luck PC, Freier T, Steudel C, Knirel YA, Luneberg E, Zahringer U, Helbig JHA (2001) Point mutation in the active site of Legionella pneumophila O-acetyltransferase results in modified lipopolysaccharide but does not influence virulence. Int J Med Microbiol 291:345–352. Moffat JF, Edelstein PH, Regula DP Jr, Cirillo JD, Tompkins LS (1994) Effects of an isogenic Zn-metalloprotease-deficient mutant of Legionella pneumophila in a guinea-pig pneumonia model. Mol. Microbiol. 12:693–705. Molofsky AB, Swanson MS (2004) Differentiate to thrive: lessons from the Legionella pneumophila life cycle. Mol. Microbiol. 53:29–40. Naylor J, Cianciotto NP (2004) Cytochrome c maturation proteins are critical for in vivo growth of Legionella pneumophila. FEMS Microbiol Lett. 241:249–256. Neumeister B, Faigle BM, Sommer M, Zähringer U, Stelter F, Menzel R, Schütt C, Northoff H (1998) Low endotoxic potential of Legionella pneumophila lipopolysaccharide due to failure of interaction with the monocyte lipopolysaccharide receptor CD14. Infect. Immun. 66:4151–4157. Pine L, Hoffman PS, Malcolm GB, Benson RF, Keen MG (1984) Determination of catalase, peroxidase, and superoxide dismutase within the genus Legionella. J. Clin. Microbiol. 20:421–429. Pine L, Hoffman PS, Malcolm GB, Benson RF, Franzus MJ (1986) Role of keto acids and reduced-oxygen-scavenging enzymes in the growth of Legionella species. J. Clin. Microbiol. 23:33–42. Rankin S, Li Z, Isberg RR (2002) Macrophage-induced genes of Legionella pneumophila: protection from reactive intermediates and solute imbalance during intracellular growth. Infect. Immun. 70:3637–3648. Retzlaff C, Yamamoto Y, Okubo S, Hoffman PS, Friedman H, Klein TW (1996) Legionella pneumophila-heat shock protein induced increase of interleukin-1␤ mRNA innvolves protein kinase C signalling in macrophages. Immunol. 89:281–288. Ristroph JD, Hedlund KW, Gowda S (1981) Chemically defined medium for Legionella pneumophila growth. J. Clin Microbiol. 13:115–119. Rossier O, Cianciotto NP (2005) The Legionella pneumophila tatB gene facilitates secretion of phospholipase C, growth under iron-limiting conditions, and intracellular infection. Infect. Immun. 73:2020–2032. Sauer JD, Bachman MA, Swanson MS (2005) The phagosomal transporter A couples threonine acquisition to differentiation and replication of Legionella pneumophila in macrophages. PNAS 102:9924–9929.


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8 Legionnaires’ Disease—Clinical Picture Thomas J. Marrie

1. Introduction The 58th annual convention of the American Legion was held at a hotel in Philadelphia from July 21 to 24, 1976 (Fraser et al 1977). Subsequently, 182 of the delegates became ill and 146 were hospitalized in 87 institutions across the United States (Tsai et al 1979). Most had radiographic evidence of pneumonia and 29 (16%) died (Fraser et al 1977). Within about six months of the outbreak a new microorganism, Legionella pneumophila, was isolated from the pulmonary tissue of some of those who died in the Philadelphia outbreak (McDade et al 1977). Over the ensuing 30 years a great deal has been learned about the clinical features of Legionnaires’ Disease (LD). In this chapter we will discuss these under the headings of epidemiology, clinical manifestations, diagnosis, and treatment.

2. Epidemiology Legionnaires’ disease may occur as sporadic cases or as outbreaks and in both instances the disease may be community or nosocomially acquired. It may also occur as a non-pneumonic form, Pontiac fever. In July 1968, an illness that involved a short incubation period, a high attack rate of 95%, with fever, headache, myalgia, and malaise. Just over half, 57%, had cough but no pneumonia occurred in 144 affected people in a new county health department building in Pontiac, Michigan (Meyer 1983; Glick et al 1978). Other symptoms included dizziness, painful stiff neck, nausea, chest pain, joint pains, sore throat, sore eyes, abdominal pain, confusion, coryza, photophobia, diarrhea, poor coordination, anorexia, nose bleed, vomiting, insomnia, bizarre dreams, rash, and irritability (Glick et al 1978). The acute illness lasted 2–5 days, although many experienced lassitude afterwards. L. pneumophila was recovered from condenser water and specimens of serum from patients showed seroconversion to Legionella. The evaporative condenser discharge units of the air conditioner had leaked into air ducts and L. pneumophila was recovered from the water (Meyer 1983).

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The first sporadic case of L. pneumophila occurred in 1947 in a ricksettsial research worker who had bacteremia (Myer 1983). The earliest known outbreak of LD occurred in 1965 at St. Elizabeth’s hospital in Washington DC. (Meyer 1983). Outbreaks have varied in size from a few cases to the largest outbreak yet reported of 800 suspected and 449 confirmed cases in Murcia, Spain, in July 2001 (Garcia-Fulgueiras et al 2003). A recent study from Europe for the period 2000–2002 indicated that there were 10,322 cases of LD with infection rates of 0 to 34.1 cases per million population among the reporting countries (Joseph 2004). From 1993 to 2000 the rate peaked at 10.1 per million in 2002. Indeed, of the 20,481 cases reported in this time frame almost half occurred in 2000–2002. Thirty-six outbreaks involving 211 persons were linked to hospitals; 38 outbreaks with 1059 cases occurred in community settings; 2 outbreaks were linked to private homes and 113 outbreaks were really travel-associated clusters involving 315 persons (Joseph 2004). Travel within Europe accounted for 88% of the cases. The remainder were associated with travel to Americas, Caribbean, Far East, Africa, and Middle East (Joseph 2004). Marston et al.(1994) analyzed Legionella surveillance data on 3254 patients reported to the CDC from 1980 through 1989. Disease rates did not vary by year but were higher in the northern states and during the summer. The mean age of patients with Legionnaires’ disease was 52.7 years compared with 34.7 years for the US population. In contrast to the earlier reports, persons with Legionnaires’ disease were now more likely to be black. They were also more likely to be smokers and to have diabetes, cancer, acquired immunodeficiency syndrome (AIDS), or end-stage renal disease. Indeed, the observed number of cases among patients with AIDS was 42-fold higher than expected. Risk factors for mortality in the study of Marston et al. (1994) were older age, male sex, nosocomial acquisition of disease, immunosuppression, end-stage renal disease, and cancer. Of the cases 23% were nosocomially acquired. Some of the features that were noted in a review of the first 1000 cases of LD in the US were 71% of the cases were male and States with the highest attack rates were east of the Mississippi River (England et al. 1981). In addition in the 2 weeks before onset of illness 37% of the patients had traveled overnight; 29% had been a hospital visitor and 5% had been hospitalized for 2 days or less before onset of illness (England et al. 1981). Other risk factors for LD in the setting of an outbreak are cigarette smoking, relative risk 1.7–3.4; consumption of 3 or more drinks of alcohol per day confers a relative risk of 3.5 (Broome and Fraser 1979). More recently, investigators have begun to combine studies of traditional risk factors with a dissection of host susceptibility using molecular biology tools. Hawn et al (2003) noted that a mutation leading to a stop codon at position 392 resulted in a dysfunctional TLR (toll like receptor) 5 protein unable to recognize flagellin was a risk factor for Legionella pneumophila infection. Interestingly this only appeared to be a risk factor in nonsmokers. At the cellular level type I interferon protects permissive macrophages from Legionella pneumophila infection through an inferferon gamma-independent pathway (Schiavoni et al. 2004), and reduced interferon gamma release has been

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noted in patients who have recovered from Legionnaires’ disease (Lettintga 2003). It is no surprise then that treatment with a tumor necrosis antagonist can predispose to Legionnaires’ disease (Wondergem et al. 2004). It is interesting in light of the above information about the role of cell-mediated immunity in LD that patients with human immunodeficiency virus (HIV) infection who have a defect in cell-mediated immunity are relatively infrequently infected with Legionella spp. However, when they do develop Legionella infection these patients take longer to become afebrile, have more respiratory symptoms, and a higher rate of respiratory failure as well as mortality when compared with patients with Legionella but without HIV infection (Pedro-Bodet et al 2003). From 1980 through 1998 there was a change in the methods of diagnosing LD in the US with a decline in the number of cases diagnosed by culture and direct fluorescent antibody test and serology and an increase in the number of cases diagnosed by the detection of antigen in the urine. These trends were associated with a decrease in the mortality rate from 26 to 10% for community-acquired cases and from 46 to 14% for nosocomial cases (Benin et al 2002). Legionellosis is believed to occur worldwide, but data are limited or non-existent for many countries. It is likely that legionellosis is uncommon in areas without hotwater heaters and complex water distribution systems. However, even in these areas, aspiration of contaminated natural water, as, for example, following boating accidents, can result in Legionnaires’ disease. Legionnaires’ disease has been found throughout North America, Europe, the United Kingdom, Argentina and Brazil in South America, Singapore, Thailand, and Australia. A few cases of Legionnaires’ disease have been reported from India. Bhopal (1993) feels that much but not all of the variation in the geographic incidence of Legionnaires’ disease is an artifact due to differences in definition, diagnostic methods, surveillance systems, and data presentation. However, in areas where studies have been carried out, Legionella and Legionnaires’ disease have been found. This situation is exemplified by data from Singapore. There were 144 cases of Legionnaires’ disease from 1991 to 1995, and there was widespread contamination of water (33% of 2774 cooling towers; 18.8% of 16 outdoor fountains) by Legionella in that country (Committee on Epidemic Diseases 1996). As with most infectious diseases, outbreaks provide an opportunity to learn about the mechanisms of transmission of Legionnaires’ disease. In most instances, Legionella is transmitted to humans by inhalation of aerosols containing the bacteria. Outbreaks have been associated with exposure to a variety of aerosolproducing devices, including showers (Breiman et al. 1990; Hanrahan et al. 1987), a grocery store mist machine (Mahoney et al. 1992), cooling towers (Dondero et al 1980; Klaucke et al 1984; Addiss et al 1989; Mitchell et al 1990; O’Mahaney et al 1990; Garbe et al 1985), whirlpool spas (Anonymous 1994; Vogt et al 1987), decorative fountains, and evaporative condensers (Cordes et al 1980; Breiman et al 1990). It is also likely that aspiration of contaminated potable water by immunosuppressed patients is also a mechanism whereby Legionella is acquired (Marrie et al 1991; Cameron et al 1991). Table 8.1 summarizes information on a number of outbreaks that have been reported.

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Country

Confirmed cases

1. Fraser et al., 1976 (July) 1977

USA (Philadelphia)

LD

182

2. Mangione et al., 1985 3. Manolen et al 1993

1982 (May)

USA (Michigan)

PF

11

1987 USA (Vermont) (October)

LD

17

4. Goldberg et al., 1989

Dec 1987– Jan 1988

Scotland (Lochgoilhead)

PF

60

USA (California)

LD

6

USA (California)

PF

36

USA (Louisiana)

LD

33

5. Breiman et al., 1990

1998 (June– July) 6. Fenstersheib 1988 et al., 1990

7. Mahoney et al., 1992

1989 (Oct– Nov) 1991 (Jan)

8. Thomas USA (Vermont) LD / et al., 1993 PF 9. Watson 1989 England (London) LD et al., 1994 (Jan–Feb) 10. Keller et al., 1993 (Sept) USA LD 1996 (Massachusetts)

6 33 11

11. Jernigan et al., 1996

1994 (April– July**)

USA

LD

16

12. Health et al., 1998 13. Fiore et al., 1998

1995 (Jan)

Australia (Sydney) LD

11

1995 (July)

USA (Pennsylvania)

LD

20

14. Kool et al., 1998 15. Luttichau et al., 1997

1996 (Sept)

USA (Missouri)

LD

3

1995

Denmark

PF

13

16. Brown et al., 1994 (July– 1999 Sept) 17. Benkel et al., 1996 (Oct) 2000

USA (Delaware)

LD

29

USA (Virginia)

LD

23

18. DeSchrijver et al., 2000

Belgium

LD

43

1999 (Nov)

Isolate

Source of outbreak

L. pneumophila airborne?

54.7 (mean) range: (3–82)

L. pneumophila Whirlpool serogroup 6 spa L. pneumophila Potable N/A serogroup 1 water in two lodges L. micdadei Whirlpool age 32 spa at (2–72) hotel L. pneumophila Condenser 77 (mean) serogroup 1 range: (56–89) L. anisa Water Two grps fountain median in hotel ages 36 lobby and 46 L. pneumophila Grocery 64 serogroup 1 store (36–88) mister L. pneumophila Whirlpool serogroup 1 L. pneumophila Cooling 15% serogroup 1 towers (5/33) L. pneumophila Cooling 59 (mean) serogroup 1 towers range: (40–74) L. pneumophila Whirlpool 62.5 serogroup 1 spas at (15–83) a flower show L. pneumophila Cooling serogroup 1 tower L. pneumophila Hospital 70.5 serogroup 1 cooling towers L. pneumophila Sump serogroup 1 pump L. pneumophila Suspect. N/A serogroup 1; WhirlL.micdadei pool L. pneumophila Cooling 53.5 serogroup 1 tower (29–80) L. pneumophila Whirlpool 66 serogroup 1 span in (42–86) store Whirlpool 49 or (mean) fountain (2–84) in a fair tent

Mortality

Date

Age (median/range in years)

Author

Presentation

TABLE 8.1. Selected outbreaks of Legionnaires’ Disease ( LD) and Pontiac Fever (PF).

16% (29/182)

18%

67% (4/6)

none

27% (3/11) 0.5% (1/50)

27% (3/11) 10% (2/20)

None

7% (2/29) 9% (2/23)

5.6 % (5/90)

8. Legionnaires’ Disease—Clinical Picture 19. Formica et al., 2000 20. Benin et al., 2002 21. Den Boer et al., 2002

1998 (Oct) 1999 (May)

Australia (Victoria) USA (Georgia)

LD

18

1999 (Feb 19– Mar 21)

Netherlands

LD / PF LD

22. Fernandez et al., 2002

1999–2000

Spain (Alcoy)

LD

177

23. GarciaFulgueiras et al. 2003

2001 Spain (June 26– July 19)

LD

449

24. Greig et al., 2004

2000 (April) Australia (Melbourne)

LD

125

25. Goeller et al., 2005

Oct 2003– Feb 2004

LD

7

USA (Maryland)

2—LD / 22—PF 133

L. pneumophila Cooling 50 serogroup 1 towers L. pneumophila Whirlpool serogroup 6 in hotel L. pneumophila Whirlpool 66 yrs serogroup 1 spas at (20–91) a flower show L. pneumophila Two 65.3 serogroup 1 different (mean cooling ⫹/ towers ⫺ 16.5) L. pneumophila Cooling 19–90 serogroup 1 tower (70%⬎ at city 50) hospital L. pneumophila Cooling 64 serogroup 1 towers (23–89) at aquarium L. pneumophila Potable 63 serogroup 1 water at (37–70) a hotel

137 none

11% (21/188)

6.2% (11/177)

1.1% (6 deaths) 3.20%

None

Legionella longbeachae accounts for 72% of the cases of LD in Western Australia (Cameron et al 1991). In this area, cases have occurred in active gardeners and the organism has been isolated from potting soil (Cameron et al 1991; Speers and Tribe 1994). An outbreak of Legionnaires disease at a flower show in the Netherlands taught us additional lessons. One-hundred-and-eighty-eight people became ill and 133 had confirmed LD (Den Boer et al 2002). There were 77,061 visitors to the flower show. The attack rate for visitors was 0.23% while for exhibitors it was 0.61%. The source was two contaminated whirlpool spas in halls 3 and 4 (Den Boer et al 2002). It is noteworthy that the whirlpools were new and were installed only 4 days previously. Also 16% of those with LD had an incubation period of more than 10 days. This large outbreak allowed investigators to determine risk factors for intensive care unit (ICU) admission and for mortality (Lettinga et al 2002). The overall mortality rate was 13%, but for those who required ICU admission it was 36%. Smoking, temperature ⱖ 38.5 C, and bilateral infiltrates on chest radiograph were independent risk factors for ICU admission or death. Institution of adequate antimicrobial therapy within 24 hours of admission was associated with higher ICU free survival rate compared with therapy initiated after 24 hours – 78% vs 54%.

3. Clinical Features of Legionnaires’ Disease A listing of symptoms and signs in patients with LD does not accurately convey the clinical features of the disease. While, as will be subsequently described, there are no features of LD that allow one to make the diagnosis clinically, there is

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little doubt from clinical observations that in young adults with no comorbidity who develop rapidly progressive pneumonia LD has to be near the top of the differential diagnosis. In the Philadelphia outbreak (Tsai et al 1979) fever was present in 97%; malaise in 89%; cough in 86%; chills 74%; dyspnea 59%; myalgias 55%; headache 53%; chest pain 52%; sputum production 50%; diarrhea 41% at presentation. Sixty percent had a white blood cell count ⬎ 10,000 per mm3 and 34% had bilateral pulmonary infiltrates on chest radiograph. When patients with LD are compared with those with community-acquired pneumonia due to other agents the patients with LD are more likely to have myalgias, headache, diarrhea, and a higher mean oral temperature at the time of presentation (Marrie et al 2003). They also present to hospital sooner after the onset of symptoms—4.7 days vs. 7.7 days (p 0.02). When patients with LD were compared with patients with bacteremic pneumococcal pneumonia, the following features were associated with Legionella pneumonia: male sex odds ratio (OR) 4.6 95% confidence interval ( CI) 1.48–14.5; heavy drinking 4.8 (1.39–16.42); previous ␤-lactam therapy 19.9 (3.47–114.2); axillary temperature ⬎ 39°C 10.3 (2.71–38.84); myalgias 8.5 (2.35–30.74); gastrointestinal symptoms 3.5 (1.01– 12.18). Negative associations included pleuritic chest pain, previous upper respiratory tract infection, and purulent sputum (Fernandez-Sabe et al 2003). In a study comparing the radiographic features of LD, pneumococcal pneumonia, mycoplasma pneumonia, and psittacosis, Macfarlane et al. (1984) noted that radiographic deterioration was a particular feature of LD occurring in 30/46 (65%) compared with 14/27 (51%) in patients with bacteremic pneumococcal pneumonia occurring. Residual intrapulmonary streaky opacities remained in over a quarter of survivors from LD and bacteremic pneumococcal pneumonia (Macfarlane et al 1984). About half the patients with LD have unilateral pneumonic involvement throughout the course of their illness (Kroboth et al 1983). The lower lobes are involved most commonly and pleural effusions are seen in about 35% (Kroboth et al 1983). The severity of the radiographic findings correlated significantly with the presence of L. pneumophila in sputum by direct fluorescent antibody. Lung abscess (Dowling et al 1983), empyema (Randolph and Beekman 1979), and bulging fissure sign are other radiographic features that are occasionally seen in patients with LD. Occasionally Legionella can be cultured for a prolonged period of time, despite treatment (Glaser et al 2005). When this occurs it is usually in an immunocompromised host (Tan et al, 200l; Schindel et al 2000); however, it has also been reported in an immunocompetent host (Glaser et al 2005). Mulazimoglu et al. (2001) critically reviewed the literature and concluded that Legionnaires’ disease cannot be diagnosed on the basis of clinical features. However, the community-based pneumonia incidence study group devised a scoring system wherein points were assigned for various degrees of temperature, serum creatinine level, serum sodium level, serum LDH, headache, vomiting, and smoking (Fernandez-Sabe et al 2003). The maximum possible score is 16. A score of ⱖ10 indicates high probability of LD, 5–9 moderate and ⱕ4 low.


139

They tested this scoring system in a case-control study with 81 cases of Legionnaires’ disease and 136 cases of pneumococcal pneumonia (FernandezSabe et al 2003). The physicians were able to identify 52/81 (64%) cases as LD and 8/136 (6%) cases of pneumococcal pneumonia as LD (Fernandez-Sabe et al 2003). The sensitivity of their scoring system was 96%, specificity 17%, positive predictive value 55%, and negative predictive value 26%. The Winthrop University Hospital scoring system devised by Cunha is based on similar premises (Cunha 1998). Gupta et al (2001) evaluated the Wintroph University Hospital scoring system and noted a sensitivity of 78% and a specificity of 65%. Relative bradycardia is seen in about two-thirds of patients with LD (Meyer 1983). Most patients appear acutely ill. Crackles are generally present on auscultation of the chest. One of the remarkable things about Legionnaires’ disease is the range of extrapulmonary manifestations. While they occur in about 30% of patients they can dominate the clinical picture and determine the outcome. There can be a variety of central nervous system manifestations, the most common of which is change in mental status. Other manifestations include lethargy, confusion, delirium, stupor, coma, seizures, hallucinations, gait disturbances, slurred speech, fine or coarse tremors, hyperactive reflexes, absence of deep tendon reflexes, signs of cerebellar dysfunction including nystagmus and gait disturbance. Peripheral neuropathy, cranial nerve palsies, including incontinence or urinary retention are other manifestations (Johnson et al. 1984; Bernardini et al 1985; Armengol et al 1992). Myocarditis, pericarditis, and endocarditis have all been reported albeit uncommonly as extrapulmonary manifestations of LD (Armengol et al 1992; Tompkins et al 1988; Svendsen et al 1987). Acute renal failure, tubulointerstitial nephritis, tubular necrosis, and rapidly progressive glomerulonephritis are the renal manifestations in LD (Poulter et al 1981; Oredugba et al 1980; Wegmuller et al 1985). Reactive arthritis and osteomyelitis are uncommon manifestations (Andereya et al 2004; McCelland et al 2004) while myalgia and arthralgia during the acute phase of the infection are very common. Legionnaires’ disease usually worsens during the first week even when therapy with erythromycin has been instituted. The clinical course of LD with currently available treatments seems to be different now than what it was when erythromycin was the treatment of choice. This is exemplified by a study of 25 patients with LD who were treated with azithromycin (Plouffe et al 2003). These patients were all diagnosed by using an assay for Legionella urinary antigen and thus early diagnosis may have accounted for the very favorable outcomes. Twenty-two of twenty-three evaluable patients were cured. At the 10-day follow-up 45% had signs and symptoms while at the 4–6-week follow-up period 35% had signs and symptoms (Plouffe et al 2003). In an international study of culture-confirmed legionellosis in 508 patients with sporadic community-acquired legionellosis, Legionella pneumophila serogroup

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1 accounted for 84% of legionellosis cases (Yu et al 2002). There are 15 serogroups of Legionella pneumophila. Legionella longbeachae accounted for 3.9% of the isolates; L. bozemanii 2.4%; L. micdadei 0.6%; L. dumoffi 0.6%; L. feeleii 0.4%; L. wadsworthii 0.2%; L. anisa 0.2%; and unknown species 0.2% (Yu et al 2002).

4. Diagnosis of Legionella Infection 4.1. Routine Laboratory Data Leucocytosis is present in up to 75% of patients (Meyer 1983). Leukopenia, thrombocytopenia and disseminated intravascular coagulopathy occur occasionally (Meyer 1983). Gram staining of expectorated sputum or endotracheal aspirates shows a few polymorphonuclear leucocytes. Organisms are usually not seen using Gram’s stain. Giminez stain does show bacilli (Meyer 1983). Microscopic hematuria is seen in about 16% of patients and proteinuria is not uncommonly seen (Meyer 1983). Hyponatremia occurs in about half the patients and is likely due to inappropriate antidiuretic hormone secretion (Meyer 1983). Liver enzymes are often mildly elevated although on occasion hepatic alkaline phosphatase can be markedly elevated and such elevation may persist (Meyer 1983).

4.2. Specific Tests The specific laboratory tests available for the diagnosis of Legionnaires’ disease include culture of blood and sputum; detection of organisms in respiratory secretions by a direct fluorescent antibody test; detection of Legionella antigen in urine specimens; antibody determination in serum samples; and detection of Legionella DNA in respiratory secretions by polymerase chain reaction. There have been two recent reviews of the laboratory diagnosis of LD (Waterer et al 2001; Den Boer et al 2004). Sputum and blood culture are 100% specific but ⬍10% sensitive. The DFA technique detects Legionella in 33–66% of cases in which sputum is obtained and is 99–100% sensitive. One of the major problems, however, is collecting an adequate sputum specimen. In general, only about one-third of patients with pneumonia are able to produce a proper sputum specimen. Detection of legionella antigen in urine is quick with a turnaround time of about one hour. It is 80–90% sensitive and 98–100% specific. Unfortunately it is only readily available for detection of L. pneumophila serogroup 1. Also it should be noted that the antigen test positivity rate varies with the severity of the disease being positive in 40–53% of mild cases and 88–100% of severe cases (Waterer et al 2001). Serology is 60–80% sensitive and 95–99% specific. One of the difficulties with serology is that acute and convalescent phase samples are necessary.


141

The convalescent sample should be collected no sooner than 6 weeks after the acute phase and indeed seroconversion has not occurred in some documented cases of LD even out to 12 weeks (Waterer et al 2001). Recent studies have shown that a previously accepted diagnostic criterion of a single antibody titer of ⱖ1: 256 is not valid (Plouffe et al 1995). A fourfold rise in antibody titer between the acute and convalescent phase samples is good evidence that infection with Legionella spp. has occurred. A not unexpected effect of the availability of urinary antigen tests for the diagnosis of LD is that over the period 1980–1998 diagnosis using this modality increased from 0 to 69% and diagnosis using culture, direct fluorescent antibody, and serology decreased markedly (Benin et al. 2002). The mortality rate for both community-acquired and nosocomial LD decreased from 34 to 12% (Benin et al. 2002), most likely because of the identification of milder cases of disease. Also the frequency of isolates other than L. pneumophila serogroup 1 decreased from 38 to 4% (Benin et al. 2002).

5. Treatment In the original outbreak of LD it was observed that those who were treated with the macrolide erythromycin had a lower mortality rate than individuals who were treated with other antibiotics (Fraser et al 1997). Subsequently, a newer macrolide, azithromycin, was shown to have a bactericidal effect in the guinea pig alveolar macrophage model and it had a 5-day post-antibacterial effect when it was removed from the system (Dedicoat and Venkatesan 1999) while erythromycin in the same model was bacteriostatic and had no post-antibacterial effect. These observations have been confirmed in clinical trials wherein 20 of 21 patients treated with azithromycin were cured (Plouffe et al 2003). It is noteworthy that since beta-lactam antibiotics do not penetrate into cells they are ineffective in Legionnaires’ disease even though they show in vitro activity (Dedicoat and Venkatesan 1999; Amsden 2005). Data from a prospective, nonrandomized study indicate that levofloxacin is superior to macrolides for the treatment of severe LD (Blazquez Garrido et al 2005). In this study carried out in Murcia, Spain, 3.4% of the patients receiving levofloxacin had complications compared with 27.2% of those receiving macrolides; the levofloxacin patients had a shorter length of stay: 5.5 vs 11.3 days. Addition of rifampin to levofloxacin provided no additional benefit. Mykietiuk et al (2005) selected 139 cases of L. pneumophila pneumonia from a prospective series of 1934 consecutive cases of community-acquired pneumonia. The overall mortality rate was 5%. Eighty patients received initial therapy with a macrolide and 40 with levofloxacin. Patients who received levofloxacin had a faster time to defervescence 2 vs 4.5 days and to clinical stability 3 vs 5 days. The complication rates were the same in both groups at 25%. The case fatality rate for those treated with levofloxacin was 2.5% vs 5% for those treated with macrolides (p ⫽ .906).

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The median length of stay was 8 days for the levofloxacin treated group and 10 days for those who received macrolides p ⫽ 0.014. In a review of data from six clinical trials, Yu et al (2004) noted that 75 patients with Legionella infection had been treated with levofloxacin. Ninety percent of these infections had resolved clinically at the post-therapy visit, 2–14 days after termination of treatment. They concluded that treatment with 500 mg of levofloxacin daily for 7–14 days or 750 mgs daily for 5 days was effective. In a study of 33 patients admitted to an ICU with LD fluoroquinolone administration within 8 hours of ICU arrival was associated with decreased mortality (Gacouin et al 2002). This is not surprising in that there are now several studies showing that administration of antibiotics to elderly patients with community-acquired pneumonia with 4–8 hours of presentation to an emergency room is associated with lower mortality than administration of the first dose of an antibiotic at a later time (Meehan et al 1997; Houck et al 2004). Many factors should be considered when deciding on the duration of therapy. Immune status and severity of the infection are probably the two most important factors. In mild to moderate cases in immunocompetent subjects with a rapid response to therapy a duration of 10 days is sufficient (Roig and Rello 2003). Indeed in this setting a 5-day course of azithromycin is probably sufficient. In patients with severe disease and/or immunocompromised a 3-week course of treatment with either fluroquinolones or macrolides (other than azithromycin) is necessary to avoid relapse (Roig and Rello 2003). One should also remember that in some patients with legionellosis, polymicrobial infection may be present (Roig and Rello 2003). Dual infection with Legionella and the following organisms have been documented—Streptococcus pneumoniae, Proteus mirabilis, Staphylococcus aureus, Escherichia coli, Prevotella intermedia, Enterococcus faecium, Enterobacter cloacae, Klebsiella pneumoniae, Haemophilus influenzae, Neisseria meningitides, Streptococcus mitis, Listeria monocytogenes, Nocardia species, Mycobacterium tuberculosis, Aspergillus, Cryptooccus, Pneumocystis jiroveci (Roig and Rello 2003). There is evidence from studies of community-acquired pneumonia that dual infection is a risk factor for more severe disease (Guiterrez et al 2005). Patients who are seriously ill with Legionnaires’ disease require management in an ICU. In this setting (sepsis and septic shock) there is evidence that lowdose corticosteroid therapy is beneficial to those who are relatively adrenal insufficient (⬍9 mcg/ml response in cortisol level to a dose of adrenocorticotrophic hormone) (Vincent et al 2002). It is also possible that activated protein C may be helpful although specific trials have not been carried out in Legionnaires’ disease (Vincent et al 2002). Hyperglycemia has been shown to be associated with higher mortality rates in patients who require hospitalization for community-acquired pneumonia so it is likely that control of hyperglycemia will be beneficial to patients with Legionnaires’ disease and elevated blood sugar (McAllister et al 2005).


143

6. Pontiac Fever In addition to the symptoms described earlier in this chapter an outbreak of Pontiac Fever among workers performing high-pressure cleaning at a sugar-beet processing plant gives additional insights into this illness (Castor et al 2005). Fourteen of the fifteen workers affected in this outbreak were using water to clean an evaporator vessel. What was different about this outbreak was that 13 had bronchoconstriction with respiratory compromise necessitating nebulizer therapy. Oxygen was administered to 12 and all received antibiotic and corticosteroid therapy. Thirteen were admitted to hospital and one was admitted to intensive care. The pronounced respiratory findings seen in this outbreak are not typical of Pontiac Fever. Four seroconverted to L. pneumophila. Very high levels of endotoxin were found in the water from the pump house and the power washers. There are two theories to explain Pontiac Fever—one is that it results from exposure to nonviable Legionella organisms and the other is that hypersensitivity to amoebal pathogens that serve as natural hosts for Legionella species may also be an etiologic factor for Pontiac Fever (Rowbotham 1986). Both theories are compatible with negative cultures and urine antigen assays in cases of Pontiac Fever. Table 8.1 lists some of the outbreaks of Pontiac Fever that have been reported. Pontiac Fever has been caused by L. pneumophila serogroup 1 (Luttichau et al 1998), L. pneumophila serogroup 6 (Mangione et al 1985), L. micdadei (Goldberg et al 1989), and L. anisa (Fenstersheib et al 1990). In one outbreak of Pontiac Fever at a hotel L. micdadei, L. dumoffi, L. maceachernii were isolated from cultures of the spa filter backwash but seroconversion occurred only to L. micdadei (Huhn et al 2005). Whirlpools at spas or hotels seem to be the main sources for outbreaks of Pontiac Fever (Table 8.1). In one outbreak due to L. anisa the mean incubation time was 49 hours and the mean duration of illness was 71 hours (Jones et al 2003). In almost every outbreak of Pontiac Fever the attack rate has been very high (Goldberg et al 1992).

7. Nosocomial Legionnaire’s Disease The clinical features of nosocomial Legionnaires’ disease are often colored by the co-morbid illness that these hospitalized patients have. However, careful study of cases of nosocomial LD reveals that 20% have lobar pneumonia; 20% have a rapidly progressive pneumonia; 16% present as aspiration pneumonia; 30% have a non-specific pattern on chest radiograph; and some cases clinically mimic pulmonary embolism (Marrie et al. 1992). The mortality rate is quite high in nosocomial LD ranging from 25 to 70% (Marrie et al. 1992). The epidemiology of nosocomial LD is dominated by contaminated water in the hospital plumbing system or through exposure to contaminated cooling towers (Guiguet et al 1987). Eradication of Legionella from the water has led to cessation of cases of nosocomial LD (Stout et al 1982). Based on this clear association the Centers for Disease Control has issued guidelines for how to eradicate

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Legionella from the water by either super heating or hyper-chlorination (Tablan et al. 2004). Occasionally other sources of nosocomial LD such as contaminated transesophageal esophageal echocardiography probes have been reported (Levy et al 2003). Nosocomial LD has also been reported in long-term care facilities and pediatric hospitals (Sennivasan et al. 2005; Campins et al 2000).

8. Summary Legionnaires’ Disease which came to our attention in dramatic fashion in 1976 remains a most fascinating disease. It can occur in outbreaks or as sporadic cases both in the community and nosocomially. Transmission is through inhalation of contaminated aerosols or through aspiration of contaminated water. The methods of diagnosis and treatment have changed over the years. Now testing of urine for Legionella antigen is commonly used but culture of respiratory secretions and amplification of Legionella DNA from these specimens are to be encouraged. Quinolones and newer macrolides are adequate for the treatment of LD. The mortality from this disease is decreasing probably because of earlier detection due to the widespread availability of the urinary antigen test but also probably because of better antibiotics and a higher index of suspicion on behalf of clinicians. Diagnosis of a case of LD should always raise the question—what is the source and the local Department of Health should be notified as one case may be the start of an outbreak.

References Addiss DG, Davis JP, LaVenture M, Wand PJ, Hutchinson MA, McKinney RM (1989) Community-acquired Legionnaires’ disease associated with a cooling tower: Evidence of longer-distance transport of Legionella pneumophila. Am J Epidemiol 130:557–568. Amsden G (2005) Treatment of Legionnaires’ disease. Drugs 65:605–614. Andereya S, Schneider U, Siebert CH, Wirtz (2004) Reactive knee and ankle joint arthritis: abnormal manifestation of Legionella pneumophila. Rheumatol Internat 24:182–184. Anonymous (1994) Outbreak of pneumonia associated with a cruise ship MMWR 43:521. Armengol S, Domingo Ch, Mesalles E (1992) Myocarditis: a rare complication during Legionella infection. Internat J Cardiol 37:418–420. Benin AL, Benson RF, Arnold KE, Fiore AE, Cook PG, Williams LK, Fields B, Besser RE (2002) An outbreak of travel associated Legionnaires disease and Pontiac fever: The need for enhanced surveillance of travel associated legionellosis in the United States. J Infect Dis 185:237–243. Benin AL, Benson RF, Besser RE (2002) Trends in Legionnaires’ disease 1980–1998: Declining mortality and new patterns of diagnosis. Clin Infect Dis 35:1039–1046. Benkel DH, McClure EM, Woolard D, Rullan JV, Graysion BM, Jenkins SR, Hershey JH, Benson RF, Pruckler JM, Brown EW, Kolczak MS, Hackler RL, Rouse BS, Breiman RF (2000) Outbreak of Legionnaires’ disease associated with a display whirlpool spa. Int J Epidemiol 29:1092–1098.


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Bernardini DL, Lerrick KS, Hoffman K, Lange M (1985) Neurogenic bladder. New clinical findings in Legionnaires’ disease. Am J Med 78:1045–1046. Bhopal RS (1993) Geographical variation of Legionnaires’ Disease: A critique and guide to future research. International J Epidemiol 22:1127–1136. Blazquez Garrido RM, Parra FJE, Frances LA, Guevara RMR, Sanchez-Nieto JM, Hernandez MS, Martinez JAS, Huerta FH (2005) Antimicrobial chemotherapy for Legionnaires’ disease; Levofloxacin versus macrolides. Clin Infect Dis 40:800–806. Breiman RF, Cozen W, Fields BS, Mastro TD, Carr SJ, Spika JS, Mascola L (1990) Role of air-sampling in investigation of an outbreak of Legionnaires’ disease associated with exposure to aerosols from an evaporative condenser. J Infect Dis 161:1257–1261. Breiman RF, Fields BS, Sanden GN, Volmer L, Meirer A, Spika JS (1990) Association of shower use with legionnaires’ disease: Possible role of amoebae. JAMA 263:2924–2926. Broome CV, Fraser DW (1979) Epidemiological aspects of Legionellosis. Epidemiological Revs 1:1–16. Brown CM, Nuorti PJ, Breiman RF, Hathcock AL, Fields BS, Lipman HB, Llewellyn GC, Hofmann J, Cetron M (1999) A community outbreak of Legionnaires’ disease linked to hospital cooling towers: an epidemiological method to calculate dose of exposure. Int J Epidemiol 28:353–359. Cameron S, Walker C, Roder D, Feldheim J (1991) Epidemiological characteristics of Legionella infection in South Australia: Implications for disease control. Aust NZ J Med 21:65–70. Campins M, Ferrer A, Callis L, Pelaz C, Cotes PJ, Pinart N, Vaque J (2000) Nosocomial Legionnaires’ disease in a children’s hospital Pediatr Infect Dis J 19:228–234. Castor ML, Wagstrom EA, Danila RN, Smith KE, Naimi TS, Besser JM, Peacock KA, Juni BA, Hunt JM, Kirkhorn S, Lynfield R (2005) An outbreak of Pontiac Fever with respiratory distress among workers performing high-pressure cleaning at a sugar-beet processing plant. J Infect Dis 191:1530–1537. Committee on Epidemic Diseases: Legionellosis surveillance in Singapore, 1991–1995 (1996) Epidemiol News Bull 22:13. Cordes LG, Fraser DW, Skaliy P, Perlino CA, Elsea WR, Mallinson GF, Hayes PS (1980) Legionnaires’ disease outbreak at an Atlanta, Georgia, country club: Evidence for spread from an evaporative condenser. Am J Epidemiol 111:425–431. Cunha BA (1998) Clinical features of Legionnaires’ Disease. Semin Respir Infect 13: 116–127. Dedicoat M, Venkatesan P (1999) The treatment of Legionnaires’ disease. J Antimicrob Chemother 43:747–752. Den Boer JW, Yzerman EPF, Schellekens J, Lettinga KD, Boshuizen HC, Van Steenbergen JE, Bosman A, Van den Hof S, Van Vilet HA, Peeters MF, Van Ketel RJ, Speelman P, Kool JL, Conyn-Van Spaendonck MAE (2002) A large outbreak of Legionnaires’ disease at a flower show in the Netherlands. Emerg Infect Dis 8:37–43. Den Boer JW, Yzerman EPF (2004)Diagnosis of Legionella infection in Legionnaires’ disease. Eur J Clin Microbiol Infect Dis 23:871–878. Dondero TJ Jr, Rendtorff RC, Mallison GF, Weeks RM, Levy JS, Wong EW, Schaffner W (1980) An outbreak of Legionnaires’ disease associated with a contaminated airconditioning cooling tower. N Engl J Med 302:365–370. DeSchrijver K, Van Bouwel E, Mortelmans L, Van Rossom P, De Beukelaer T, Vael C, Dirven K, Goossens H, Leven M, Ronveaux O (2000) An outbreak of legionnaires’ disease among visitors to a fair in Belgium in 1999. Eurosurveillance 5:115–119.

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Greig JE, Carnie JA, Tallis GF, Ryan NJ, Tan AG, Gordon IR, Zwolak B, Leydon JA, Guest CS, Hart WG (2004) An outbreak of Legionnaires’ disease at the Melbourne aquarium, April 2000: Investigation and case-control studies. Med J Austral 1980:566–572. Guiguet MJ, Pierre J, Burn P, Berthelot G, Gottot S, Gibert G, Valleron AJ (1987) Epidemiological survey of a major outbreak of nosocomial legionellosis. Int J Epidemiol 16: 466–471. Guiterrez F, Masia M, Rodriguez JC, Mirete C, Soldan B, Padilla S, Hernandez I, Royo G, Martin-Hidalgo A (2005) Community-acquired pneumonia of mixed etiology, prevalence and outcome. Europ J Clin Microbiol Infect Dis 24:377–383. Gupta SK, Imperiale TF, Sarosi GA (2001) Evaluation of the Wintrop University Hospital criteria to identify Legionella pneumonia. Chest 120:1064–1071. Hanrahan JP, Morse DL, Scharf VB, Debbie JG, Smith JP, McKinney RM, Shayegani M (1987) A community hospital outbreak of legionellosis: Transmission by potable hot water. Am J Epidemiol 125:639–649. Hawn TR, Verbon A, Lettinga KD et al (2003) A common dominant TLR 5 stop codon polymorphism abolishes flagellin signaling and is associated with susceptibility to legionnaires’ disease. J Exp Med 198:1563–1572. Heath TC, Roberts C, Jalaludin B, Goldthrope I, Capon AG (1998) Environmental investigation of a legionellosis outbreak in western Sydney: the role of molecular profiling. Aust N Z J Public Health 22:428–431. Houck PM, Bratzler DW, Nsa W et al (2004) Timing of antibiotic administration and outcomes for Medicare patients hospitalized with community-acquired pneumonia. Arch Intern Med 164:637–644. Huhn GD, Adam B, Ruden R, Hilliard L, Kirkpatrick P, Todd J, Crafts W, Passaro D, Dworkin MS (2005) Outbreak of travel-related Pontiac fever among hotel guests illustrating the need for better diagnostic tests. J Travel Med 12:173–179. Jernigan DB, Hofmann J, Cetron MS, Genese CA, Nuorti JP, Fields BS, Benson RF, Carter RJ, Edelstein PH, Guerrero IC, Paul SM, Lipman HB, Breiman RF (1996) Outbreak of Legionnaires’ disease among cruise ship passengers exposed to a contaminated whirlpool spa. Lancet 347:494–499. Johnson JD, Raff MJ, Van Arsdall JA (1984) Neurologic manifestations of Legionnaires’ disease. Medicine 63:303–310. Jones TF, Benson RF, Brown EW, Rowland JR, Crosier SC, Schaffner W (2003). Epidemiologic investigation of a restaurant-associated outbreak of Pontiac fever. Clin Infect Dis 37:1292–1297. Joseph CA. Legionnaires’ disease in Europe 2000–2002 (2004) Epidemiol Infect 132:417–424. Keller DW, Hajjeh R, DeMaria A, Fields BS, Pruckler JM, Benson RS, Kludt PE, Lett SM, Mermel LA, Giorgio C, Breiman RF (1996) Community outbreak of Legionnaires disease: An investigation confirming the potential for cooling towers to transmit Legionella species. Clin Infect Dis 22:257–261. Klaucke DN, Vogt RL, LaRue D, Witherell LE, Orciari L, Spitalny KC, Pelletier R, Cherry WB, Novick LF (1984) Legionnaires’ disease: The epidemiology of two outbreaks in Burlington, Vermont, 1980.Am J Epidemiol 119:382–391. Kool JL, Warwick MC, Pruckler JM, Brown EW, Butler JC (1998) Outbreak of Legionnaires’ disease at a bar after basement flooding. Lancet 351:1030. Kroboth FJ, Yu VL, Reddy SC, Yu AC (1983) Clinicoradiographic correlation with the extent of Legionnaires’ disease. Am J Radiol 141:263–268.

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Levy PY, Teysseire N, Etienne J, Raoult D ( 2003) A nosocomial outbreak of Legionella pneumophila caused by contaminated transesophageal probes. Infect Control Hosp Epidemiol 24:619–622. Lettinga KD, Verbon A, Weverling G-J, Schellekens JFP, Den Boer JW, Yzerman EPF, Prins J, Boersma WG, van Ketel RJ, Prins JM, Speelman P (2002) Legionnaires’ Disease at a Dutch flower show: Prognostic factors and impact of therapy. Emerging Infect Dis 8:1448–1454. Luttichau HR, Vinther C, Uldum SA, Muller J, Faber M, Jensen JS (1997) An outbreak of Pontiac fever among children following a use of a whirlpool. Clin Infect Dis 26:1374–1378. Macfarlane JT, Miller AC, Smith WHR, Morris AH, Rose DH (1984) Comparative radiographic features of community acquired legionnaires’ disease, pneumococcal pneumonia, mycoplasma pneumonia, and psittacosis. Thorax 39:28–33. Mahoney FJ, Hoge CW, Farley TA, Barbaree JM, Breiman RF, Benson RF, McFarland LM (1992) Community-wide outbreak of Legionnaires’ disease associated with a grocery store mist machine. J Infect Dis 165:736–739. Mangione EJ, Remis RS, Tait KA, McGee HB, Gornan GW, Wentworth BB, Baron PA, Hightower AW, Barbaree JM, Broome CV (1985). An outbreak of Pontiac fever related to whirlpool use, Michigan 1982. JAMA 253:535–539. Manolen M, Breiman RF, Barbaree JM, Gunn RA, Stone KM, Spika KS, Dennis DT, Mao SH, Vogt RL (1993) Use of multiple molecular subtyping techniques to investigate a Legionnaires disease outbreak due to identical strains at two tourist lodges. J Clin Microbiol 31:2584–2588. Marrie TJ, De Carolis E, Yu VL, Stout J and the Canadian Community-acquired Pneumonia Investigators (2003) Legionnaires’ disease—results of a multicenter Canadian study. Can J Infect Dis 14:154–158. Marrie TJ, Haldane D, Bezanson G (1992) Nosocomial Legionnaires’ disease: clinical and radiographic patterns. Can J Infect Dis 3:253–260. Marrie TJ, Haldane D, MacDonald S, Clarke K, Fanning C, Le Fort Jost S, Bezanson G, Joly G (1991) Control of nosocomial Legionnaires’ disease by using sterile potable water for high risk patients. Epidemiol Infect 107:591–605. Marston BJ, Lipman HB, Breiman RF (1994) Surveillance for Legionnaires’ disease. Risk factors for morbidity and mortality. Arch Intern Med 154:2417–2422. McAllister F, Rowe BH, Majumdar SR, Romney J, Blitz S, Marrie TJ ( 2005) The relationship between hyperglycemia and outcomes in 2,471 patients admitted to hospital with community-acquired pneumonia. Diabetes Care 28:810–815. McClelland MR, Vaszar LT, Kagawa F (2004) Pneumonia and osteomyelitis due to Legionella longbeachae in a woman with systemic lupus erythematosus. Clin Infect Dis 38:102–106. McDade JE, Shepard CC, Fraser DW, Tsai TR, Redus MA, Dowdle WR, and the Laboratory Investigation Team ((1977)) Legionnaires’ Disease: Isolation of a bacterium and demonstration of its role in other respiratory diseases. N Engl J Med 297:1197–1203. Meehan TP, Fine MJ, Krumholz HM, Scinto JD, Galusha JM, Mockalis JT, Weber GF, Petrillo MK, Houck PM, Fine JM (1997) Quality of care, process, and outcomes in elderly patients with pneumonia. JAMA 278:2080–2084. Meyer RD (1983) Legionella Infections: A review of five years of research. Rev Infect Dis 5:258–278. Mitchell E, O’Mahoney M, Watson JM, Lynch D,Joseph C, Quigley C, Aston R, Constable GN, Farrand RJ, Maxwell S (1990) Two outbreaks of Legionnaires’ disease in Bolton Health District. Epidemiol Infect 104:159–170.


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9 Legionella pneumophila: Innate and Adaptive Immunity Herman Friedman, Catherine Newton and Thomas Klein

1. Introduction Legionella pneumophila is a ubiquitous opportunistic pathogen which frequently causes serious infection in humans, especially immunocompromised individuals in institutions such as nursing homes or hospitalized patients receiving chemotherapy for organ transplantation or cancer. In general, most normal individuals are resistant to this microorganism, which has as its natural habitat warm water containing amoeba, protozoa or other single cell organisms14,15,18,34,39,50,51. Legionella readily replicate in human monocytes, especially macrophages. Infection of human or susceptible animal macrophages/monocytes results in rapid growth of the bacteria28,43. Generally within 48–72 hrs of culture there is a 100- to 1000-fold increase in Legionella number in infected cells. This rate of multiplication in macrophages is much more rapid than for other opportunistic bacteria which replicate intracellularly, such as mycobacteria or salmonellae47,58–60,62. There is now much information how legionellae attach to and enter into a host cell like a macrophage or a single cell protozoa, including signal transduction induced by adherence, uptake, entry mechanisms and initial intracellular survival1,3,4,8,9,11,13,17,22,26. Most adults have been exposed to L. pneumophila and have serum antibody titers to these bacteria greater than 1:200 by the second decade of life without apparent clinical disease6,12,16,18,20,21,26,48. Furthermore, most normal individuals readily develop antibody and cell mediated immune responses to these Gram-negative bacteria without overt clinical symptoms19,56. Extensive studies after the initial isolation and recognition of these organisms three decades ago showed that both crude and purified L. pneumophila antigens are stimulatory for immune cells in vitro, as well as for susceptible animals, including guinea pigs14,15,19. However, most mouse strains are resistant, except A/J mice which are genetically more susceptible58,61. Several studies have shown that a host response to Legionella infection involves both cellular and humoral aspects of immunity. Humans usually develop a vigorous immune response following infection by the respiratory route, which may result in acute pneumonia20,21. Although the mechanisms of immune resistance to Legionella are not completely understood, it is widely recognized that cell mediated immune (CMI) 151

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responses depending on phagocytic cells such as macrophages and dendritic cells, as well as lymphocytes, have an important role in suppressing growth and eliminating Legionella from the host. Immunocompromised individuals, including those with an immunodeficiency disease such as AIDS, are deficient in lymphocytes which functionally respond to the microbe14,15,20.

2. Legionella Immunogens Results of many studies concerning immune resistance to Legionella since their discovery and recognition in the mid-1970s showed that several distinct aspects of both innate and adaptive immune responses are important for host defense and share many features in common with immunity to other intracellular opportunistic microorganisms. Studies during the past few decades provide much information concerning prevention and treatment of individuals infected by these bacteria, which still account for about 25,000 reported cases each year in the USA alone14. However, correct and prompt diagnosis of Legionella infection and use of antibiotics prevent widespread acute pneumonia cases, as occurred in Philadelphia in 1976 when several thousand Pennsylvania State American Legion veterans attended their annual convention in a center city hotel and over 200 developed acute disease, with about 25 deaths18. These Gram-negative bacteria contain many antigens, including lipopolysaccharide (endotoxin), heat shock and outer membrane proteins, flagella and pili23,27,32,52. These components are strong stimulators of immunity, including production of antibodies, cytokines and chemokines16,18. Fig. 9.1 is a schematic presentation of L. pneumophila antigens which stimulate cytokines and humoral factors, including antibodies. These immunogenic components directly stimulate macrophages to produce a wide variety of cytokines, including colony stimulating factor and interleukins, especially IL-1 (Fig. 9.2). The proinflammatory cytokine tumor necrosis factor (TNF␣), in particular, is rapidly stimulated in macrophages infected with virulent Legionellae. Although less toxic and pyrogenic than enterobacterial endotoxins, L. pneumophila endotoxin is a potent stimulator of proinflammatory cytokines

Legionella pneumophila Legionella pneumophila Hsp60 Hsp60 LPS

FIGURE 9.1. Schematic representation of L. pneumophila antigens which stimulate innate and adaptive immunity induced by cytokines and immune cells.

Omps Omps

MSP

Cytokines

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Adaptive Immunity

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FIGURE 9.2. Macrophages stimulated by L. pneumophila, especially components such as HSP, produce the cytokine IL-1.

Heat shock proteins

IL – 1

PKC

Macrophages

produced by immune cells, including both macrophages/dendritic cells and lymphocytes23,30,40,42,51,52. Serum from patients with active Legionella infection or following recovery have antibodies not only to flagella and pili, but also to structural components of the bacteria, including heat shock proteins (HSPs). Although HSPs are located predominantly in the intracellular compartment of most bacteria, they are expressed on the surface of L. pneumophila, as well as secreted extracellularly27. Therefore, involvement in immune stimulation is evident. For example, L. pneumophila HSPs readily induce cytokine production by macrophages. In addition, HSPs increase the steadystate level of various cytokine messages, including those for alpha/beta interferons (IFN), IL-1, IL-6, TNF␣ and GM-CSF (Fig. 9.3). Increases in cytokine activity occur through a cell surface receptor system involving protein kinase C dependent pathways. HSPs as well as other Legionella components such as major outer membrane protein (MOMP) are dominant in activating T lymphocytes and also stimulating antibody responses during infection16,18,54. Although in experimental animals HSPs or other components, as well as non-viable heat-killed Legionella, have the potential to serve as a protective vaccine, they are only minimally effective in clinical trials. In contrast, animal studies demonstrated good immune responses in vivo to both whole bacteria and components. The results of guinea pigs and mice studies show that antigenic components (MOMP) or killed Legionella vaccines stimulate both innate and adaptive immune responses16,18,20,54,29–31.

3. Innate Immunity to L. pneumophila The course of infection in susceptible experimental animals, such as genetically susceptible mice, can be divided into an early phase during which there is rapid bacterial multiplication and an inflammatory response. A second phase begins

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Antimicrobial activity

IFNγ

Th1

Cytokines (IL–12)

Th0

FIGURE 9.3. L. pneumophila infection stimulates cytokine production involved in both innate and acquired immune responses and initiates a cascade of immune factors.

about 2–3 days after infection with downregulation of the non-specific response, resulting in a decrease in the number of bacteria in infection sites, especially the lungs. This early cellular response is characterized by an interstitial inflammatory reaction consisting mainly of macrophages, B lymphocytes and natural killer (NK) cells, as well as immature dendritic cells (DCs)16,18. These lymphoid cells represent a first line of defense against infection and represent innate immunity triggered by bacterial surface antigens (Figure 9.3). Factors involved in innate immunity include macrophage activation by the cytokine IFN␥, production of the proinflammatory cytokine TNF␣ and cytokines IL-6 and IL-1, as well as chemokines from inflammatory cells4,21,31,32,34,50. Mobilization of these cytokines are protective, but when high levels are induced and become excessive, toxic shock-like death may occur32,33,41. In the case of human infection, the initial phase of Legionnaires’s disease in susceptible individuals is development of overt symptoms corresponding to an acute phase reaction due to proinflammatory cytokine mobilization53,57. Innate immune responses to L. pneumophila infection limits the growth of the bacteria, which occurs mainly in macrophages. Regulation of bacterial replication in target macrophages by proinflammatory cytokines, especially TNF␣, appears to be the major event in the early phase of infection4,20. However, other cytokines are also produced by macrophages and NK cells involved in innate immunity to Legionella (Fig. 9.4). In this regard, endogenous IFN␥ production by NK cells is important in regulating L. pneumophila growth. However, dendritic cells in experimental animals genetically susceptible to L. pneumophila are not as readily infected in vitro as macrophages and this does not appear related to interferon35.

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There are distinctions between innate and adaptive immunity relating to welldefined cytokines and chemokines. TNF␣ produced mainly by macrophages is a potent cytokine that affects regulation of many immune and inflammatory cells. This cytokine enhances anti-bactericidal activity of macrophages to many intracellular microbes, including L. pneumophila37,38. Both the murine system and studies with human peripheral blood lymphocytes show that both IFN␥ and TNF␣ are involved in Legionella growth inhibition not only in macrophages but also in neutrophils treated with these cytokines20. Furthermore, TNF␣ and IFN␥ are present in bronchial secretions after respiratory infection of experimental mice with L. pneumophila6. Treatment of the animals with exogenous TNF␣ results in significant protection from mortality, probably by neutrophil function activation. TNF␣ also enhances phagocytic activity and oxidative bursts of human neutrophils and facilitates resolution of infection mediated in part by nitric oxide. As is evident in Fig. 9.4, many cytokines are produced by Lp-infected macrophages important for innate immunity. Some cytokines important in antimicrobial immunity include not only TNF␣, but also IFN␥ produced by lymphocytes, including NK cells. TNF␣ produced by macrophages/DCs activated lymphocytes evince increased bactericidal activity. For example, addition of exogenous TNF␣ to human peripheral blood monocytes in vitro inhibits bacterial replication and mediates resistance of monocytes to L. pneumophila infection. In addition, anti-TNF␣ antibody treatment of the cells in vitro increases their susceptibility to infection. Although TNF␣ has an important role in innate immunity to L. pneumophila infection mediated by phagocytic cells, including neutrophils, cytokines such as IL-12 produced by dendritic cells and macrophages after Legionella infection stimulate Th1 helper cells involved in adaptive immunity.

TNFα TNF IL-1 GM-CSF

IL-6 MIP-2

IFN γ

MIP-1 IL-12

NK

TNFα

PMN TNFα increases bactericidal activity

FIGURE 9.4. Activation of macrophages/ DCs for innate immunity to L. pneumophila, especially TNF␣ and IFN␥ production as well as PMN antibacterial activity.

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It should be noted that non-phagocytic cells, including NK cells, in addition to killing tumor and other foreign cells, are recognized as an important early source of cytokines. For example, L. pneumophila induces production of IFN␥ in cultured splenocytes and NK cells after stimulation with killed bacterial antigens. Chemokines are known also to have a crucial role in mobilization and activation of various immune cell types to inflammatory sites. Chemokines are responsible for rapid migration of leukocytes from the circulation, serving a critical role in innate immunity to bacterial infection. L. pneumophila infection of macrophages in vitro induces production of several important chemokines, including macrophage inflammatory protein 1␤ (MIP-1␤), MCP-2 and KC20,21. Monocyte chemotactic protein 3 (CCL7) gene expression is rapidly induced in macrophages infected with virulent but not avirulent L. pneumophila43. In contrast, other chemokine genes are induced by both virulent and avirulent Legionella. Such findings indicate that innate immune responses, at least in regards to chemokine induction by L. pneumophila infection, depend on the virulence of the infecting bacterium. Such a regulated response of innate immunity to microbial pathogens may contribute to host homeostasis, since over production of cytokines/chemokines is harmful. Although chemokine induction mechanisms in bacterial infection remain unclear, such induction appears regulated by receptors/ligands distinguishable from other cytokine inducible systems. Differential regulation of chemokine vs. cytokine induction indicates that innate immunity related to proinflammatory mediators like chemokines vs. certain cytokines such as IL-1 and GM-CSF is regulated differently, probably through different bacterial receptors.

4. Adaptive Immunity to L. pneumophila Infection The adaptive immune response to bacterial infection follows initial exposure to microbes, not necessarily active infection, and is transferable to naïve recipients by lymphocytes. The humoral antibody response to bacterial infection is a typical adaptive immune response when an individual is exposed to bacterial antigens and a heightened specific antibody response then occurs after secondary stimulation by antigens from the same microbe or active infection. The role of antibodies in resistance to infection varies according to the bacterium. In the case of Legionella infection, various specific virulence factors and specific components or antigens have been elucidated20. However, the importance of these antigens for the humoral immune response in humans has not yet been clearly defined. In contrast, the role of many immune factors involved in Legionella immunopathogenesis in experimental animals has been characterized. Antibodies to Legionella antigens and other components promote uptake and replication of the bacteria in macrophages rather than promoting microbe elimination55,49. Therefore, antibodies and humoral immunity to Legionella are thought to have a minimal role in resistance or as a second line of defense to infection The role of cellular adaptive immunity to Legionella infection, however, is critical. Delayed type hypersensitivity (DTH) reactions and CMI activity of


157

IL-18 Cell-mediated Immunity

DC 1 /APC

NK

IL-12

Th1 IF N

Bacteria

IL-12

γ

CD4

IL-2 IFNγ IgG2a

Th0 CD4 IL-4

Th2 CD4

CD4

2

DC

IFNγ

IL-4 IL-5 IL10 IgG1 Antibody Immunity

FIGURE 9.5. Th1 and Th2 helper cell activity stimulated by DCs and APC. Solid lines indicate promotion of cell function and dotted lines indicate inhibition.

lymphoid cells from either immunized animals or patients recovered from legionellosis are readily detected. For example, peripheral blood lymphocytes from recovered patients, as compared to non-infected controls, show a marked proliferative response when cultured with L. pneumophila48. After stimulation with Legionella, peripheral blood cells from recovered patients secrete immunoregulatory cytokines which restrict growth, indicative of CMI. In particular, Th1-defining cytokines like IFN␥ and IL-12, which restrict Legionella growth, are elevated in blood cell culture supernatants from recovered patients. Furthermore, animal studies show that both Th1 and Th2 helper cell defining cytokines develop after Legionella infection in vivo (Fig. 9.5). It seems evident that Th1 and Th2 helper cell development is under the control of antigen-recognizing cells like DCs and antigen presenting cells (APCs). As evident in Fig. 9.5, Th1 CD4⫹ cells are stimulated to produce proinflammatory cytokines such as IL-2 and IFN␥ important in CMI. Th2 CD4⫹ cells produce IL-4, IL-5 and IL10 and these are involved in humoral (antibody) immunity.

5. Immune Mechanisms In terms of mechanisms involved, most conclusive studies have been performed with genetically susceptible mice. Macrophages from susceptible A/J strain mice readily replicate these bacteria in culture21,58. In contrast, macrophages from genetically resistant BALB/c or C57BL/6 strain mice are less permissive for

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intracellular growth of these organisms in culture. However, in vivo susceptibility to challenge infection with Legionella is only moderately different (less than one log) between susceptible vs. resistant strains. For example, genetically susceptible A/J mice are only moderately more susceptible than resistant BALB/c or C57BL/6 mice. An immunosuppressive drug like cyclosporine markedly suppresses T-lymphocyte function and the reconstitution of such immunosuppressed mice with T cell culture supernatants containing Th1 cytokines increases resistance, indicating that lymphocytes as well as Th1 cytokines like IFN␥ are important for host resistance mediated by macrophage activation. Thus, a vital role for lymphocytes in controlling L. pneumophila infection seems apparent, at least in animal models. Many studies show the importance of CMI in bacterial infections depends on differentiation of T helper cells phenotypes. The functions of Th1 vs. Th2 cells correlate with the type of cytokine produced. For example, Th1 cytokines activate cytotoxic and inflammatory functions and therefore these cells are recognized as important in cell mediated inflammatory reactions. In contrast, Th2 cytokines are associated with strong antibody responses and, therefore, involved in humoral immunity. These cytokines also cross-regulate the activity of each T helper cell phenotype. The Th1 cytokine IFN␥ inhibits proliferation of Th2 cells while the Th2 cytokine IL-10 inhibits functions of Th1 cells. This cross-regulation apparently explains the strong bias of Th1 responses against intracellular microbial infection. In general, Th1 cells are more likely to mediate protection against intracellular pathogens like Legionella whereas extracellular microbes appear to be better controlled by combinations of Th2 and Th1 type cytokines. Experimental infection of mice with L. pneumophila results in immunologic features common to most intracellular bacterial infections. These include activation of Th1 cells and other characteristic CMI responses in mice sublethally infected with L. pneumophila, which induces specific resistance to secondary infection with a lethal challenge of the bacteria45. Splenocytes from primed mice are sensitized and proliferate to a greater extent and produce more IFN␥ when stimulated in vitro with Legionella. Furthermore, challenge re-infection of primed mice results in a marked increase in anti-Legionella responses, including a simultaneous increase in splenic CD4⫹ and CD8⫹ T cells. IL-12, which induces Th1 cell activity, appears early in the serum post-infection (Fig. 9.6) followed by IFN␥, as well as serum immunoglobulin G2. Enhanced production of Th1 cytokines during L. pneumophila infection in humans also has been reported. Significant increases of IFN␥ and IL-12 levels occur during the acute phase of infection, but Th2 cytokines IL-4 and IL-10 are usually not increased in Legionella infected patients. Serum IL-12 levels remain high or increase further during convalescence. It is evident that after infection with L. pneumophila, serum levels of IL-12 p40/p70 increases within hours and then decreases rapidly thereafter (Fig. 9.6). Thus, the relative predominance of Th1 related cellular immune responses is evident during legionellosis. Involvement of Th1 phenotypes and their immune products, as well as Th2 cytokines such as IL-4 or IL-10, during Legionella infection is considered


FIGURE 9.6. Increases in serum IL-12 p40/p70 after L. pneumophila infection.

24 20 IL-12 (ng/ml)

159

16 12 8 4 0

1

3

5 8 10 12 16 24 Hours after Infection

complex44. An elevated IL-4 level in infected mice is induced early after infection (i.e., 3 hrs), but does not interfere with development of the protective Th1 response. Rapid appearance of IL-4 in serum and in ex vivo splenocytes cultures from infected mice occurs only during the early phase of infection (Fig. 9.7). It is evident that L. pneumophila infection induces a rapid rise in IL-4 production with a peak level at 3–4 hrs after infection and a rapid decline thereafter, whereby at 24 hrs ex vivo spleen cell cultures from infected mice show essentially no IL-4 production. An increase in IL-4 response to Legionella infection is also evident at the mRNA level. As evident in Fig. 9.8, there is a rapid upregulation of IL-4 message shortly after L. pneumophila infection, followed by a rapid downregulation. Furthermore, IL-4 knockout mice are more susceptible to L. pneumophila infection, indicating this cytokine has a protective role (Table 9.1). In addition, IL-4 knockout mice evince elevated levels of TNF␣, IL-1b and IL-6, and exogenous IL-4 treatment of L. pneumophila-infected macrophage cultures suppresses

∗

250

IL-4 (pg/ml)

200

∗

150 100 50 0 N

3 5 24 Hours after Infection

FIGURE 9.7. IL-4 production induced by L. pneumophila infection. Spleen cells from Legionella-infected mice produce IL-4 in ex vivo culture at various times, with peak responses of 3 and 5 hrs.

160


1.20

A ∗

1.00

IL-4/β-actin duplex

∗ 0.80

∗

∗ ∗

0.60

0.40

0.20

0.00

N

0.5

1 2 3 5 8 16 Time (hr) post infection (PI) BALB/c

FIGURE 9.8. Upregulation of IL-4 mRNA in ex vivo splenocyte cultures early after L. pneumophila infection followed by rapid downregulation. Data represent 5–10 mice per column and means ⫾ SEM, p ⬍ 0.05. *Significant

compare to control cultures from non-infected mice.

production of these cytokines. Treatment of IL-4 knockout mice with anti-TNF␣ antibody protects against L. pneumophila infection. The IL-4 knockout mice also develop robust IFN␥ production to the bacteria and survive a secondary challenge infection with lethal numbers of Legionella. Thus IL-4 has an important regulatory function and downregulates acute cytokines which otherwise would be induced by L. pneumophila infection44. It is clear the control of differentiation of uncommitted T cell precursors to Th1 vs. Th2 cells is at least partially regulated TABLE 9.1. L. pneumophila-induced mortality in IL-4 competent and deficient BALB/c mice. L. pneumophila infection dose 2 ⫻ 107 107 7 ⫻ 106 5 ⫻ 106 3 ⫻ 106 106

Dead/total (% mortality) BALB/c Mice (wt) (IL-4tm2Nnt) 5/5 (100) – 3/5 (60) – 0/12 (0) 6/6 (100) – 6/8 (75) – 1/5 (20) – 0/3 (0)


161

DC

Jagged1 ICD

Notch receptor

RBPJκ

ICN

repressor

Th2 GATA3 IL-4

activator

FIGURE 9.9. NOTCH 1 signaling pathway in Th2 cells. Jagged 1 on DCs bind to NOTCH 1 on T cells activating intracellular domain and cellular proteins converting a repressor to activator to initiate GATA-3 and IL-4 transcription.

by cytokines (e.g., IL-4 stimulates differentiation into Th2 cells whereas IFN␥ and IL-12 enhances Th1 development). Therefore, a strong bias toward Th1 differentiation, supported by IFN␥ and IL-12 produced by macrophages/dendritic cells and/or NK cells, occurs as a result of acute Legionella infection and also during the convalescent phase. Th2 cell maturation involves signaling through NOTCH receptors (Fig. 9.9). In addition, expression of GATA-3, a zinc finger protein, is an important transcription factor and its expression is required for Th2 cell development. GATA-3 knockout mice have defective T cell development. CD4⫹ T cells express different NOTCH receptors and antigen presenting cells such as macrophages and DCs express various NOTCH ligands. The Jagged ligand binds to NOTCH receptors (Figure 9.9). Delta4 induces Th1 responses. Jagged1 expression is needed for IL-4 production. In addition, different NOTCH receptors are important for different responses. However, the mechanism concerning involvement of NOTCH in polarization of T cells is not completely understood. For example, blocking NOTCH signaling in CD4⫹ cells does not prevent developing a protective Th2 response, but there is still a normal Th1 response. On the other hand, inhibiting expression of NOTCH receptor activity by gamma-secretase inhibitors prevents T-bet gene activation and Th1 activity. It is important to understand the role of NOTCH receptors and ligands expressed on T cells and DCs in T helper cell polarization for infectious diseases, including legionellosis. We have shown that Legionella infection of DCs induces expression of both Jagged1 and Delta4 (Fig. 9.10), possibly accounting for the T helper cell activation induced by Legionella. Studies concerning modulation of innate immunity also permit a better understanding of the role of DCs in resistance to intracellular opportunistic bacteria like Legionella.

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FIGURE 9.10. Increase of Jagged1 and Delta 4 message in L. pneumophila-infected DCs. Cells infected in vitro and RNA message determined 24 hrs later.

DC

LpDC

Jagged1 1.1*

1.8

0.3

0.7

Delta4

β-actin * Target to β – actin ratio

Microbial antigens stimulate antigen-presenting cells (APCs) through pattern recognition receptors like toll-like receptors (TLRs) and stimulation of these receptors results in APCs polarizing changes affecting T helper cell development2,5,7. The expression patterns of TLRs on APCs induced by various immune stimulants are quite complex. L. pneumophila LPS and viable L. pneumophila function through TLR2 and not TLR4 as would expected2,7,23. However, TLR2deficient mice control the infection by producing proinflammatory cytokines/ chemokines, recruitment of neutrophils, and clearance of L. pneumophila, while MyD88 deficient mice were susceptible to pneumonia24. Also, Archer and Roy demonstrated with intranasal-infected MyD88- and TLR2-deficient mice on permissive lgn1 allele background that MyD88 deficiency, but not TLR2 deficient mice, succumb to the L. pneumophila infection5. Several groups have demonstrated the importance of flagella and its receptor, TLR5, or flagellin in L. pneumophila infection10,42,49. In humans, a common polymorphism in the TLR5 gene causes a deficiency in mediating signals from flagellin that was associated with increased susceptibility to Legionnaires’ disease25. DC cultures infected with Legionella produce increased levels of the Th1 polarizing cytokine IL-12p4030. Legionella, like other microorganisms, directly induce DCs to produce these cytokines by interaction with receptors of pattern recognition that respond to a variety of pathogen-associated molecular patterns. When stimulated, DCs may also produce a variety of other cytokines7,36. TLR 9 may be involved in Legionella-induced production of IL-12, since recent studies showed that TLR9 is involved in IL-12 production induced by the bacterium like Brucella29. We have investigated the possible role of TLR 9 in Legionella immunity46. In addition to the role of TLR 2 and TLR4, TLR 9 mRNA by DCs was evident from Legionella resistant BALB/c mice. The CD 11c cells were about 90% positive for TLR 9 shown by specific binding of the TLR 9 ligand ODN 1826FITC. Macrophages from Legionella-resistant BALB/c mice were also 90% positive for TLR 9 determined by specific ligand binding. A requirement for TLR 9 activation is endosomal maturation and/or acidification, which is inhibited by chloroquine pretreatment. To determine whether Legionella-stimulated IL-12 production is through TLR 9, DCs and macrophages from susceptible A/J mice were stimulated in a recent study with formalin-killed


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Legionella, control E. coli LPS, ODN 1826 or infected with virulent Legionella, either in the presence or the absence of chloroquine. Chloroquine attenuated the ODN 1826 stimulation of IL-12p40 and also suppressed this cytokine production in response to either killed or viable Legionella, indicating IL-12 was stimulated through TLR 9. Similar results with chloroquine occurred with BALB/c mouse DCs. Chloroquine treatment did not affect the uptake and intracellular survival of Legionella, since colony forming units of the bacteria were similar for DCs from both mouse strains and macrophages from A/J mice. The number of L. pneumophila increased rapidly in infected A/J mouse macrophages, but intracellular growth did not occur in DCs from either mouse strain. In addition to suppression by chloroquine, activation of TLR 9 was assessed by treatment with the ligand inhibitor ODN2088. Treatment with this inhibitor completely attenuated IL-12 p40 production in response to the TLR 9 ligand ODN 1826 in DCs from both BALB/c and A/J mice (Fig. 9.11). Thus TLR 9 is important in the response of DCs as well as macrophages to Legionella. Other studies showed that DCs from TLR 4-deficient mice have a robust IL-12 p40 response after infection with live Legionella while DCs from TLR 2-deficient mice are deficient for IL 12 production. These data suggest that TLR2 and 9, but not TLR4, are mediating the DC IL 12 response to Legionella infection. T helper cell polarization is also regulated by chemokines. For example, Legionella infection of murine DC cultures increased the Th1 chemokines CCL5 (RANTES), CCL4 (MIP-1␤) and CCL3 (MIP-1␣) but not the Th2 chemokine CCL11. For these experiments 5 ⫻ 106 murine bone marrow-derived DCs were infected at a ratio of 10:1 bacteria to DCs. The DCs responded to Legionella by interacting with signal transduction molecules, proinflammatory cytokines and their receptors, as well as chemokines and their specific receptors.

70

IL-12p40 (ng/ml)

IL-12p40 (ng/ml)

60 50 40 30

C) 100

Lp ODN2088+Lp

B) 3.0 ODN1826 ODN2088 +ODN1826 2.5 2.0 1.5 1.0

*

20 0.5 10 0

* 1.0

0.0

kLp ODN2088+kLp

80 IL-12p40 (ng/ml)

A) 80

* 0.2

* 40 20

* *

* 0.5

60

0 1.0

0.2

0.5

1.0

DC Concentrations x 106

FIGURE 9.11. TLR plus inhibitor ODN 2088 induces IL-12 production in DCs infected with L. pneumophila. (A) TLR 9 ligand ODN 1826 plus 2088 inhibitor, (B) DCs pretreated with or without inhibitor and infected with live Lp, (C) DCs pretreated with or without inhibitor stimulated with killed Lp. IL-12 determined by ELISA at 24 hrs.

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FIGURE 9.12. Enhanced survival of Legionella-infected mice immunized with Lp-infected DCs vs. control non-infected DCs (70% vs. none) 24–48 hrs after infection. (ten mice per group).

DC

100

LpDC

90 80

Survival (%)

70 60 50 40 30 20 10 0

0

12

24

36 48 Time (h)

60

72

Recent studies show that antigen-loaded DCs may be useful as a powerful immunogenic vaccine (Fig. 9.12). Bone marrow-derived DCs infected in vitro with relatively small numbers of Legionella can be used as a potent vaccine to immunize syngeneic naive mice to evince immune resistance to otherwise lethal challenge with viable Legionella. For example, mice injected weekly 2–3 times with untreated DC cultures are uniformly susceptible to challenge Legionella infection, succumbing within 24–48 hrs to the bacteria with no survivors35. In contrast, mice given three weekly injections of DCs previously infected (loaded) with Legionella had a 70% survival rate after challenge infection with lethal numbers of the bacteria. Splenocytes from the Legionella/DC immunized mice produced high levels of the Th1 cytokine IFN␥, indicating that the Legionella-infected DCs were an excellent immunostimulatory vaccine, enhancing resistance of the recipient mice to Legionella and this was related to heightened production of Th1-defining cytokines (Fig. 9.13). DC maturation markers cell surface MHC class II, CD 86 and CD40 were also enhanced (Fig. 9.14).

6. Discussion and Conclusions L. pneumophila is the species of the genus Legionella most commonly associated with human disease, especially pneumonia. These bacteria infect humans, susceptible animals and single cell protozoa. They are a ubiquitous intracellular opportunistic microbe widespread in the environment, especially in aquatic habitats, persisting for extended periods in single cell paramecia such as amoeba, and cause occasionally acute infection in humans, especially immunocompromised

9. Legionella pneumophila: Innate and Adaptive Immunity DC LpDC

165

10

1500

* IFNγ (ng/ml)

IL-12p40 (pg/ml)

8 1000

500

6 4 2 0

0

FIGURE 9.13. IL-12 and IFN␥ stimulation by Lp sensitized vs. uninfected CD4⫹ cells cocultured with untreated DCs or DCs pretreated with Lp. Each bar indicates cytokine activity of 3–5 culture supernates (48 hr). Mean ⫾ SD, p ⫽ ⬍ 0.05.

100

3 102 10 FL2-1A

M1

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103 102 FL2-1A

101

104

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3 102 10 FL2-CD86

M1

101

103 102 FL2-CD86

Counts 0 5 10 15 20 25

Counts 0 5 10 15 20 25 104 100

M1

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M1

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Counts 0 5 10 15 20 25

101

Counts 0 5 10 15 20 25

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M1

Counts 0 5 10 15 20 25

Counts 0 5 10 15 20 25

individuals. Host resistance to this ubiquitous microbe is based on both innate and adaptive immune responses. Adaptive immunity consists of interactions among activated T cells and their subsets as well as humoral factors like cytokines produced by lymphocytes and activated macrophages that restrict growth and spread of the microbe within its intracellular environment. It is known that innate immunity to microbial infections, including organisms like Legionella, is also important and complex. The innate immune system includes various host cells and cytokines and recent studies have revealed regulatory roles of IL-12, IL4 and TNF␣ in controlling the early stages of infection20,21,32. Helper T cells produce IFN, enhanced by IL-12 and IL-18. The Th2 cytokine IL-4 also is a critical regulatory cytokine. Although this cytokine mainly has differentiating activity for Th 2 cells, IL-4 regulates Th1 cell-based immune responses to an intracellular bacterium like L. pneumophila44.

104 100

3 102 10 FL2-CD40

DC 104

DC\Lp

M1

101

103 102 FL2-CD40

104

FIGURE 9.14. FACS scan of untreated (DC) vs. Legionella infected DCs (DC/Lp) assayed for surface markers MHC II (I-A), CD86 and CD40 indicating a rapid increase in DC maturation.

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Clearly immune responses to Legionella have many features in common with responses to other intracellular, Gram-negative microbes, both in experimental animals and lymphoid cells cultured in vitro from patients recovered from legionellosis. Such knowledge about these bacteria is necessary to develop strategies to protect against infection by this pathogen. Although L. pneumophila actually has as its “natural host” single cell protozoa in warm water, the microbe “accidentally” infects humans as an opportunistic pathogen, primarily by interaction with alveolar macrophages, similar to single cell protozoa. Humans appear mainly to be an inadvertent host. The immune response mechanism of most healthy individuals is usually adequate to resist clinical disease by this bacterium. Further studies concerning pathogenesis and immune responses to Legionella which preferentially infect macrophages will provide valuable new information how the immune system is activated against similar intracellular pathogens.

7. Summary Legionella pneumophila is a ubiquitous opportunistic bacterium which may cause serious infection in humans, especially immunocompromised individuals, often resulting in life-threatening pneumonia. Legionella antigens stimulate the immune response, both humoral and cellular, in experimental animals and humans, resulting in both innate and adaptive immune responses involved in host resistance. Most adults, even without clinical evidence of infection, evince serum antibodies to these ubiquitous bacteria. In addition, peripheral blood lymphocytes from normal individuals respond to Legionella antigens by lymphoproliferation. Patients recovered from Legionella infection especially have strong secondary immune responses to the microbe, including higher antibody titers. Peripheral blood cells from normal and convalescent patients after stimulation with Legionella antigen also produce immunoregulatory cytokines. Furthermore, studies with experimental animals, especially susceptible as well as resistant mouse strains, have provided new information about both innate and adaptive immunity to these bacteria. For example, infection of susceptible mice with viable Legionella results in a rapid specific immune response, both innate and adaptive. Such responses are mediated by both T and B lymphocytes, NK cells and macrophages, as well as mature dendritic cells. Macrophages, in particular, are rapidly activated by inflammatory cytokines, as well as chemokines, produced by stimulated leukocytes. Active infection by Legionella induces even higher levels of cytokines which stimulate Th1 helper cells important for cellular immunity to intracellular microbes. NOTCH receptors and ligands expressed on T lymphocytes as well as DCs are important for T helper cell polarization, i.e., Th1 vs. Th2 cells expressing distinct cytokines. In addition TLRs, especially TLR9, recognize Legionella antigens resulting in DCs producing the important cytokine IL-12. Th helper cell polarization is also regulated by chemokines induced by Legionella infection of DCs, resulting in production of enhanced Th1


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type cytokines. Furthermore, DCs loaded with small numbers of the bacteria become a potent vaccine which can immunize syngeneic mice to resist challenge infection with lethal numbers of Legionella. Splenocytes from Legionella/DC immunized mice produce high levels of the Th1 cytokines, which enhance resistance of recipient mice to Legionella. Immunity to Legionella mediated by activated T cells, monocytes/macrophages and DCs, including the cytokines they produce, inhibit growth and spread of the microorganism in competent animals. Immune studies with Legionella provide much needed information on the cellular mechanisms of this ubiquitous microbe as well as to other intracellular bacteria.

Acknowledgement. The authors of this chapter enthusiastically appreciate the excellent assistance of Izabella Perkins, Joe Chou, Tangying Lu and Raymond Widen in some of the studies described. We also gratefully acknowledge the assistance of Nicholas Burdash and Ilona Friedman in preparing this manuscript. Studies reported from the authors laboratories are supported by grants from the National Institutes of Health.

References 1. Abu-Zant, A., M. Santic, M. Molmeret, S. Jones, J. Helbig, and Y. Abu Kwaik. 2005. Incomplete activation of macrophage apoptosis during intracellular replication of Legionella pneumophila. Infect Immun 73:5339–49. 2. Akamine, M., F. Higa, N. Arakaki, K. Kawakami, K. Takeda, S. Akira, and A. Saito. 2005. Differential roles of Toll-like receptors 2 and 4 in in vitro responses of macrophages to Legionella pneumophila. Infect Immun 73:352–61. 3. Andrews, H. L., J. P. Vogel, and R. R. Isberg. 1998. Identification of linked Legionella pneumophila genes essential for intracellular growth and evasion of the endocytic pathway. Infect Immun 66:950–8. 4. Arata, S., C. Newton, T. W. Klein, Y. Yamamoto, and H. Friedman. 1993. Legionella pneumophila induced tumor necrosis factor production in permissive versus nonpermissive macrophages. Proc Soc Exp Biol Med 203:26–9. 5. Archer, K. A., and C. R. Roy. 2006. MyD88-dependent responses involving toll-like receptor 2 are important for protection and clearance of Legionella pneumophila in a mouse model of Legionnaires’ disease. Infect Immun 74:3325–33. 6. Blanchard, D. K., J. Y. Djeu, T. W. Klein, H. Friedman, and W. E. Stewart. 1987. Induction of tumor necrosis factor by Legionella pneumophila. Infect Immun 55:433–7. 7. Braedel-Ruoff, S., M. Faigle, N. Hilf, B. Neumeister, and H. Schild. 2005. Legionella pneumophila mediated activation of dendritic cells involves CD14 and TLR2. J Endotoxin Res 11:89–96. 8. Byrd, T. F., and M. A. Horwitz. 1991. Chloroquine inhibits the intracellular multiplication of Legionella pneumophila by limiting the availability of iron. A potential new mechanism for the therapeutic effect of chloroquine against intracellular pathogens. J Clin Invest 88:351–7. 9. Derre, I., and R. R. Isberg. 2004. Macrophages from mice with the restrictive Lgn1 allele exhibit multifactorial resistance to Legionella pneumophila. Infect Immun 72:6221–9.

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10. Dietrich, C., K. Heuner, B. C. Brand, J. Hacker, and M. Steinert. 2001. Flagellum of Legionella pneumophila positively affects the early phase of infection of eukaryotic host cells. Infect Immun 69:2116–22. 11. Diez, E., S. H. Lee, S. Gauthier, Z. Yaraghi, M. Tremblay, S. Vidal, and P. Gros. 2003. Birc1e is the gene within the Lgn1 locus associated with resistance to Legionella pneumophila. Nat Genet 33:55–60. 12. Edson, D. C., H. E. Stiefel, B. B. Wentworth, and D. L. Wilson. 1979. Prevalence of antibodies to Legionnaires’ disease. A seroepidemiologic survey of Michigan residents using the hemagglutination test. Ann Intern Med 90:691–3. 13. Egawa, K., T. W. Klein, Y. Yamamoto, C. A. Newton, and H. Friedman. 1992. Enhanced growth restriction of Legionella pneumophila in endotoxin- treated macrophages. Proc Soc Exp Biol Med 200:338–42. 14. Fields, B. S. 1996. The molecular ecology of legionellae. Trends Microbiol 4:286–90. 15. Finegold, S. M. 1988. Legionnaires’ disease—still with us [editorial]. N Engl J Med 318:571–3. 16. Fischer, S. F., J. Vier, C. Muller-Thomas, and G. Hacker. 2006. Induction of apoptosis by Legionella pneumophila in mammalian cells requires the mitochondrial pathway for caspase activation. Microbes Infect 8:662–9. 17. Fortier, A., E. Diez, and P. Gros. 2005. Naip5/Birc1e and susceptibility to Legionella pneumophila. Trends Microbiol 13:328–35. 18. Fraser, D. W., T. R. Tsai, W. Orenstein, W. E. Parkin, H. J. Beecham, R. G. Sharrar, J. Harris, G. F. Mallison, S. M. Martin, J. E. McDade, C. C. Shepard, and P. S. Brachman. 1977. Legionnaires’ disease: description of an epidemic of pneumonia. N Engl J Med 297:1189–97. 19. Freedman, A. P., and S. M. Katz. 1981. The prevalence of serum antibodies to Legionella pneumophila in patients with chronic pulmonary disease. Am Rev Respir Dis 123:238–9. 20. Friedman, H., Y. Yamamoto, and T. W. Klein. 2002. Legionella pneumophila pathogenesis and immunity. Semin Pediatr Infect Dis 13:273–9. 21. Friedman, H., Y. Yamamoto, C. Newton, and T. Klein. 1998. Immunologic response and pathophysiology of Legionella infection. Semin Respir Infect 13:100–8. 22. Gebran, S. J., Y. Yamamoto, C. Newton, T. W. Klein, and H. Friedman. 1994. Inhibition of Legionella pneumophila growth by gamma interferon in permissive A/J mouse macrophages: role of reactive oxygen species, nitric oxide, tryptophan, and iron(III). Infect Immun 62:3197–205. 23. Girard, R., T. Pedron, S. Uematsu, V. Balloy, M. Chignard, S. Akira, and R. Chaby. 2003. Lipopolysaccharides from Legionella and Rhizobium stimulate mouse bone marrow granulocytes via Toll-like receptor 2. J Cell Sci 116:293–302. 24. Hawn, T. R., K. D. Smith, A. Aderem, and S. J. Skerrett. 2006. Myeloid Differentiation Primary Response Gene (88)- and Toll-Like Receptor 2-Deficient Mice Are Susceptible to Infection with Aerosolized Legionella pneumophila. J Infect Dis 193:1693–702. 25. Hawn, T. R., A. Verbon, K. D. Lettinga, L. P. Zhao, S. S. Li, R. J. Laws, S. J. Skerrett, B. Beutler, L. Schroeder, A. Nachman, A. Ozinsky, K. D. Smith, and A. Aderem. 2003. A common dominant TLR5 stop codon polymorphism abolishes flagellin signaling and is associated with susceptibility to legionnaires’ disease. J Exp Med 198:1563–72. 26. Heath, L., C. Chrisp, G. Huffnagle, M. LeGendre, Y. Osawa, M. Hurley, C. Engleberg, J. Fantone, and J. Brieland. 1996. Effector mechanisms responsible for


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gamma interferon-mediated host resistance to Legionella pneumophila lung infection: the role of endogenous nitric oxide differs in susceptible and resistant murine hosts. Infect Immun 64:5151–60. Hoffman, P. S., L. Houston, and C. A. Butler. 1990. Legionella pneumophila htpAB heat shock operon: nucleotide sequence and expression of the 60-kilodalton antigen in L. pneumophila-infected HeLa cells. Infect Immun 58:3380–7. Horwitz, M. A., and S. C. Silverstein. 1980. Legionnaires’ disease bacterium (Legionella pneumophila) multiples intracellularly in human monocytes. J Clin Invest 66:441–50. Huang, L. Y., K. J. Ishii, S. Akira, J. Aliberti, and B. Golding. 2005. Th1-like cytokine induction by heat-killed Brucella abortus is dependent on triggering of TLR9. J Immunol 175:3964–70. Kikuchi, T., T. Kobayashi, K. Gomi, T. Suzuki, Y. Tokue, A. Watanabe, and T. Nukiwa. 2004. Dendritic cells pulsed with live and dead Legionella pneumophila elicit distinct immune responses. J Immunol 172:1727–34. Klein, T. W., H. Friedman, and S. Specter. 1998. Marijuana, immunity and infection. J Neuroimmunol 83:102–15. Klein, T. W., Y. Kawakami, C. Newton, and H. Friedman. 1991. Marijuana components suppress induction and cytolytic function of murine cytotoxic T cells in vitro and in vivo. J Toxicol Environ Health 32:465–77. Klein, T. W., C. Newton, R. Widen, and H. Friedman. 1993. Delta 9-tetrahydrocannabinol injection induces cytokine-mediated mortality of mice infected with Legionella pneumophila. J Pharmacol Exp Ther 267:635–40. Klein, T. W., C. A. Newton, N. Nakachi, and H. Friedman. 2000. Delta 9-tetrahydrocannabinol treatment suppresses immunity and early IFN-gamma, IL-12, and IL-12 receptor beta 2 responses to Legionella pneumophila infection. J Immunol 164:6461–6. Lu, T., C. Newton, I. Perkins, H. Friedman, and T. W. Klein. 2006. Cannabinoid treatment suppresses the T helper cell polarizing function of mouse dendritic cells stimulated with Legionella pneumophila infection. J Pharmacol Exp Ther 319:269–276. Lu, T., C. Newton, I. Perkins, H. Friedman, and T. W. Klein. 2006. Role of cannabinoid receptors in Delta-9-tetrahydrocannabinol suppression of IL-12p40 in mouse bone marrow-derived dendritic cells infected with Legionella pneumophila. Eur J Pharmacol 532:170–7. Matsunaga, K., T. W. Klein, C. Newton, H. Friedman, and Y. Yamamoto. 2001. Legionella pneumophila suppresses interleukin-12 production by macrophages. Infect Immun 69:1929–33. Matsunaga, K., H. Yamaguchi, T. W. Klein, H. Friedman, and Y. Yamamoto. 2003. Legionella pneumophila suppresses macrophage interleukin-12 production by activating the p42/44 mitogen-activated protein kinase cascade. Infect Immun 71:6672–5. McDade, J. E., C. C. Shepard, D. W. Fraser, T. R. Tsai, M. A. Redus, and W. R. Dowdle. 1977. Legionnaires’ disease: isolation of a bacterium and demonstration of its role in other respiratory disease. N Engl J Med 297:1197–203. McHugh, S. L., C. A. Newton, Y. Yamamoto, T. W. Klein, and H. Friedman. 2000. Tumor necrosis factor induces resistance of macrophages to Legionella pneumophila infection. Proc Soc Exp Biol Med 224:191–6. McHugh, S. L., Y. Yamamoto, T. W. Klein, and H. Friedman. 2000. Murine macrophages differentially produce proinflammatory cytokines after infection with virulent vs. avirulent Legionella pneumophila. J Leukoc Biol 67:863–8.

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42. Molofsky, A. B., B. G. Byrne, N. N. Whitfield, C. A. Madigan, E. T. Fuse, K. Tateda, and M. S. Swanson. 2006. Cytosolic recognition of flagellin by mouse macrophages restricts Legionella pneumophila infection. J Exp Med 203:1093–104. 43. Nakachi, N., K. Matsunaga, T. W. Klein, H. Friedman, and Y. Yamamoto. 2000. Differential effects of virulent versus avirulent Legionella pneumophila on chemokine gene expression in murine alveolar macrophages determined by cDNA expression array technique. Infect Immun 68:6069–72. 44. Newton, C., S. McHugh, R. Widen, N. Nakachi, T. Klein, and H. Friedman. 2000. Induction of interleukin-4 (IL-4) by Legionella pneumophila infection in BALB/c mice and regulation of tumor necrosis factor alpha, IL-6, and IL-1beta. Infect Immun 68:5234–40. 45. Newton, C. A., T. W. Klein, and H. Friedman. 1994. Secondary immunity to Legionella pneumophila and Th1 activity are suppressed by delta-9-tetrahydrocannabinol injection. Infect Immun 62:4015–20. 46. Newton, C. A., I. Perkins, R. H. Widen, H. Friedman, and T. W. Klein. 2006. Role of TLR9 in Legionella pneumophila-induced IL-12p40 production in bone marrowderived dendritic cells and macrophages from permissive and non-permissive mice. Infect Immun. 47. Newton, C. A., R. Widen, H. Friedman, and T. W. Klein. 1994. Lymphocyte subset changes following primary and secondary infection of mice with Legionella pneumophila. Immunology & Infectious Diseases 5:18–26. 48. Plouffe, J. F., and I. M. Baird. 1982. Lymphocyte blastogenic responses to L. pneumophila in acute Legionellosis. J Clin Lab Immunol 7:43–4. 49. Ren, T., D. S. Zamboni, C. R. Roy, W. F. Dietrich, and R. E. Vance. 2006. Flagellindeficient Legionella mutants evade caspase-1- and Naip5-mediated macrophage immunity. PLoS Pathog 2:e18. 50. Salins, S., C. Newton, R. Widen, T. W. Klein, and H. Friedman. 2001. Differential Induction of Gamma Interferon in Legionella pneumophila- Infected Macrophages from BALB/c and A/J Mice. Infect Immun 69:3605–10. 51. Sampson, J. S., B. B. Plikaytis, and H. W. Wilkinson. 1986. Immunologic response of patients with legionellosis against major protein-containing antigens of Legionella pneumophila serogroup 1 as shown by immunoblot analysis. J Clin Microbiol 23:92–9. 52. Sonesson, A., E. Jantzen, K. Bryn, L. Larsson, and J. Eng. 1989. Chemical composition of a lipopolysaccharide from Legionella pneumophila. Arch Microbiol 153:72–8. 53. Tateda, K., T. Matsumoto, Y. Ishii, N. Furuya, A. Ohno, S. Miyazaki, and K. Yamaguchi. 1998. Serum cytokines in patients with Legionella pneumonia: relative predominance of Th1-type cytokines. Clin Diagn Lab Immunol 5:401–3. 54. Weeratna, R., D. A. Stamler, P. H. Edelstein, M. Ripley, T. Marrie, D. Hoskin, and P. S. Hoffman. 1994. Human and guinea pig immune responses to Legionella pneumophila protein antigens OmpS and Hsp60. Infect Immun 62:3454–62. 55. Widen, R. H., T. W. Klein, C. A. Newton, and H. Friedman. 1989. Induction of interleukin 1 by Legionella pneumophila in murine peritoneal macrophage cultures. Proc Soc Exp Biol Med 191:304–8. 56. Widen, R. H., C. A. Newton, T. W. Klein, and H. Friedman. 1993. Antibody-mediated enhancement of Legionella pneumophila-induced interleukin 1 activity. Infect Immun 61:4027–32. 57. Winn, W. C., Jr., and R. L. Myerowitz. 1981. The pathology of the Legionella pneumonias. A review of 74 cases and the literature. Hum Pathol 12:401–22.


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10 Nonspecific Stimulation of Immunity Against Legionella James Rogers, Amal Hakki and Herman Friedman

1. Introduction Much is now known about Legionella pneumophila (Lp), including biology, habitat and physiology. The relationship between Lp and numerous other members of this class of opportunistic intracellular bacteria, which have many similar yet distinct properties from other opportunistic Gram-negative bacterial pathogens, has been elucidated in numerous studies during the last few decades. There is much information available about immunobiology, immunopathogenesis and the disease this organism causes. Host immune responsiveness to these bacteria, both innate and adaptive, is now generally understood. Clinical infection caused by these bacteria, especially atypical respiratory tract infection, such as pneumonia, results in rapidly elevated specific humoral antibody. However, without correct and specific diagnosis infection may quickly progress, since treatment delay even with an effective antibiotic is not sufficient to reverse rapid and fulminating disease. For this reason, there is much effort to develop an effective vaccine for immunization against these microbes. However, even if effective, it would be difficult to justify active vaccination of large populations of normal individuals, because major outbreaks of legionellosis are considered rare. Immunodeficient individuals, especially patients treated with an immunosuppressive antimetabolite for cancer or organ transplantation, are known to be at high risk. Thus, many such individuals are prophylactically given wide spectrum antibiotics. Susceptible individuals could be candidates for vaccination if an effective antibody specific antimicrobial vaccine were available. However, immunocompromised individuals would still be susceptible to other opportunistic but minimally pathogenic microbes, including viruses, fungi or other bacteria widely prevalent in the environment. Alternative use of prophylactic antibiotics has many drawbacks. Microbiologists and infectious disease investigators concerned with protection and defense against potential infectious agents for bioterrorism have similar concerns. Various scenarios include the possible use of aerosolized microorganisms as biologic weapons, which would have devastating effects over large areas (Alibek, 1999). Government agencies concerned with protection against a possible biologic attack assumed that infectious agents used may be undetected until victims 173

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become ill, obviously complicating diagnosis and treatment. Even once a biologic agent has been detected, additional time is necessary to identify the microorganism, further complicating diagnosis and treatment. The most likely microorganism for bioterrorism would be one that is readily aerosolized and therefore primarily causes pulmonary infection. Individuals exposed to such bacterial agents most likely would not be specifically vaccinated, since such a microbe may not be considered a public health threat or a vaccine is not available, similar to the situation with Lp. Natural products have been proposed as a potential stimulator of nonspecific innate immunity against a microbe used for possible bioterrorist attack (Masihi, 2000).

2. Stimulation of Immunity by Natural Products The immune system, including pulmonary immunity, can be nonspecifically stimulated in an antigen- independent manner by natural immunostimulators studied extensively in pre-clinical and clinical investigations. For example, nonspecific immunostimulators for treatment of bacterial infections have been reported but not extensively studied for therapeutic effects vs. traditional antibacterial therapy with antibiotics or specific protective vaccines. Nevertheless, it seems likely that stimulation of nonspecific innate immunity may be prophylactic and enhance resistance to pulmonary infections. Relatively nontoxic microbial components, as well as synthetic compounds, nonspecifically stimulate a protective antimicrobial immune system. For example, studies in the last few decades with natural products from plants resulted in nonspecific enhancement of both cellular and humoral immune responses to many microorganisms, especially innate immunity (Masihi, 2000). Additionally, various nutritional substances also have been studied in regards to increasing resistance to various microbes. Substances as diverse as those obtained from chili peppers, garlic and even honey extracts have been used for centuries as traditional remedies for infectious diseases. Similarly, a high resinous “propolis” product collected by honey bees from various plant sources has been a popular folk medicine and evinces a broad spectrum of biologic activities, including antimicrobial and anti-inflammatory properties (Banskota et al., 2001; Khalil, 2006).

3. Antibacterial and Antiviral Effects of Epigallocatechin Gallate The use of such substances to prevent or moderate infectious diseases is a cornerstone of traditional Chinese medicine and also alternative medicine in Western countries. In this regard, there has been increased interest in prophylactic treatment to prevent or ameliorate clinical infection or re-infection by opportunistic microbes which cause chronic disease, including gastritis due to H. pylori. Such substances are often derived from extracts of plants considered medicinal. In particular, many

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recent studies have been reported with green tea extracts, especially polyphenols such as catechins, which are the active component of tea (Mabe et al., 1999; Yamaguchi et al., 2002). Although many catechins are present in tea, the most common is epigallocatechin gallate (EGCG), which accounts for much of the in vitro antimicrobial activity of tea first reported in biomedical journals a century ago. Catechins (also known as flavanols) are group of polyphenols found in many natural plant-derived products, particularly green tea. The four major catechins are EGCG, epigallocatechin (EGC), epicatechin gallate (ECG), and epicatechin (EC) (Yang et al., 2002). The typical percentage of individual catechins in green tea extracts is 10–15% ECGC, 2–3% ECG, 2% EC and 2–3% EGC (Suganuma et al., 1999). Thus, EGCG is the major catechin in green tea, and also accounts for most of the reported biological effects of green tea, especially antitumor effects (Morre et al., 2003). Demonstrable antimicrobial activity by EGCG has been reported against a wide variety of microorganisms, including Chlamydia trachomatis, Chlamydia pneumoniae (Yamazaki et al., 2003) and Helicobacter pylori (Yanagawa et al., 2003). EGCG has also been considered to have an antiviral activity, as shown by its potent effect in vitro in HIV-1 (Yamaguchi et al., 2002). Furthermore, a number of laboratories reported that a variety of nonspecific cytokines induced by EGCG have direct antimicrobia1 activity against various microorganisms, including common extracellular bacteria like staphylococci, streptococci or even fungi. EGCG treatment of Lp-infected macrophages resulted in enhanced production of TNF␣, which markedly inhibited Lp virulence activity. Specifically, a report published in 2002 showed that the growth of Lp in permissive macrophages was selectively inhibited by small amounts of EGCG, the major active green tea catechin. This antimicrobial activity was not due to direct effects on the bacteria itself, since EGCG did not alter Lp growth in the incubation medium, regardless of various Lp concentrations used (Matsunaga et al., 2001a). Epigallocatechin gallate also rescued permissive macrophages from diminished resistance to Lp infection due to either cigarette smoke condensate or nicotine. Again, the mechanism by which EGCG overcame enhanced susceptibility to infection caused by treatment with these immunosuppressive substances involved, at least in part, upregulation of TNF␣, as well as IFN␥ production by activated macrophages, since neutralization of these cytokines by monoclonal antibodies markedly abolished the protective effects of EGCG (Matsunaga et al., 2002). IFN␥ produced by NK cells has also recently been reported as a key factor in controlling Lp infection in a permissive mouse model (Sporri et al., 2006).

4. Modulation of Cytokines by Natural Products The possibility that cytokines like TNF␣ and IFN␥ mediate in vitro antimicrobial effects of EGCG in permissive macrophages against Lp is plausible given the importance of such cytokines for activation of macrophages against bacterial pathogens.

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TNF␣ is known to enhance phagocytic and antimicrobial activities of macrophages. Mice lacking TNF␣ or TNF receptors are highly susceptible to intracellular infections (Plitz et al., 1999). Thus, the major green tea catechin EGCG may trigger an early innate immune response by increasing production of cytokines like TNF␣ and IFN␥, important for host defense against pathogenic microorganisms. Modulation of cytokine production appears to be a common theme with respect to natural plant-derived products long considered as having antibacterial properties. Induction of TNF␣ by plant-derived products does not appear to be a unique feature of EGCG. For example, Capsaicin (CAP), the active ingredient of hot pepper, has no effect on phagocytic activity by peritoneal macrophages from BALB/c mice, but increased TNF␣ production in groups of mice fed 20 ppm CAP compared to control (Yu et al., 1998). The garlic derivative alliin, another plant-derived natural product well known for its medicinal properties, reportedly increases pokeweed mitogen (PWM) induced peripheral blood mononuclear cell (PBMC) proliferation and TNF␣ production, as well as the engulfing capacity of phagocytic cells (Salman et al., 1999).

5. The Effects of EGCG on Cytokine Production by Legionella-Infected Dendritic Cells Dendritic cells (DCs) are also phagocytic cells which play a vital role in innate immunity. However, the main characteristic of DCs is their potent ability to take up antigens, mature by undergoing various phenotypic and functional changes such as the upregulation of co-stimulatory molecules and the induction of an adaptive immune response through interaction with T cells. Unlike bone marrow derived macrophages (BMMs), bone marrow derived dendritic cells (BMDCs) from susceptible A/J mice are resistant to Lp intracellular growth, a factor which may allow DCs ample time to present antigens for an adaptive cell-mediated immune response (Neild and Roy, 2003). Various reports showed that EGCG has pronounced effects on cytokine production by various types of immune cells. In particular, EGCG was shown to inhibit various proinflammatory cytokines of BMDCs from a resistant strain of mice (BALB/c) after infection with Lp, including the important Thl biasing cytokine IL-12 (Fig. 10.1). Interestingly, however, EGCG did not have an inhibitory effect on TNF␣ production by DCs. Instead, EGCG augmented TNF␣ production by the BMDCs in response to Lp infection (Fig. 10.1). This apparent contradiction of downregulation of an important pro-inflammatory cytokine, IL-I2, critical for cellular immunity against Lp infection (Matsunaga et al., 2001b), and upregulation of another proinflammatory cytokine, TNF␣, suggests that benefits of the natural tea catechin EGCG against these bacteria may have an enhancing effect on the innate immunity response rather than an adaptive response. However, the in vivo physiological effects of catechins on host resistance to Lp infection still remain to be elucidated.


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FIGURE 10.1. EGCG inhibits IL-12 upregulation but stimulates TNF-alpha production by Mouse Bone Marrow DCs in response to L. pneumophila (Lp) infection. Results are expressed as mean value in ng/ml ⫾ SEM from 5 independent experiments. The asterisks indicate statistically significant differences P ⬍ 0.05 from values of the nonEGCG-treated Lp infected cells. Similar results occurred when either LPS from E. coli or muramyldipeptide (MDP) was used to stimulate the DCs.

6. The Effects of EGCG on Co-stimulatory Molecule Expression by Legionella-Infected Dendritic Cells A recent study concerning the effects of Lp infection of DCs showed that EGCG treatment inhibited surface expression of co-stimulatory molecules by BMDCs from Lp-resistant BALB/c mice (Fig. 10.2). Expression of co-stimulatory molecules is a marker of DC maturation and associated with enhanced ability to activate resting T cells. Studies showed that EGCG treatment of cultured Lp-infected DCs inhibited the Lp-induced expression of both MHC class I and class II molecules important for DC maturation. Mature DCs process exogenous antigens intracellularly and present them to T cells within the context of MHC class II molecules, although most cells use MHC class I molecules to present peptides derived from endogenously synthesized proteins. However, DCs can deliver exogenous antigens directly to the MHC class I pathway, a phenomenon known as crosspresentation important for resistance of cells infected with Lp (Haycock, 1993; Heath et al., 2004). The inhibitory effects of EGCG on co-stimulatory/MHC molecule expression occurs simultaneously but to a greater extent than EGCG effects on the percentage of cells expressing the CD11c surface marker. Interestingly, exposure of DCs to Lp increased the percentage of cells expressing the surface marker CD11c, whereas untreated DCs evince the typical decreased CD11 c percentage, down to near control levels. Such results suggest that CD11c is a marker for DC maturation.

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FIGURE 10.2. EGCG inhibits Legionella pneumophila (Lp) upregulation of MHC class I/II and co-stimulatory molecule expression by DCs infected with Lp and treated with various concentrations of EGCG and analyzed by FACS. Numbers in quadrants reflect percentages rounded to next greater whole integer. Results shown are 1 of 5 independent experiments with similar results.

7. The Effects of EGCG on Chemokine Production by Legionella-Infected Dendritic Cells Maturing DCs involved in innate or adaptive immunity are also an abundant source of chemokines in a precise time-ordered fashion. After stimulation with a microbial antigen, DCs have an initial burst of MIP-la (CCL3), MIP-1b (CCL4) and IL-8 (CXCL8) production within a few hours. RANTES (CCL5) and MCP-1 are also induced but in a more steady manner. At a later time point DCs produce mainly lymphoid chemokines such as CCLl7 (TARC), CCLl8 (DC-CDI), CCLl9 (MIP-3B) and CCL22 (MDC) that attract T and B lymphocytes (McColl, 2002; Sallusto et al., 2000). A recent study by our laboratory showed that Lp induced upregulation of the early inflammatory mediators such as CCL3, RANTES and MCP-1, while EGCG effect in a dose-dependent manner inhibited the upregulation of these molecules (Matsunaga et al., 2001a). Again, the inhibitory effects of EGCG on important parameters, such as DC maturation and production of proinflammatory cytokines and chemokines, like IL-12 and MCP-1 is seemingly contradictory given reports demonstrating antimicrobial effectiveness of EGCG. Perhaps the answer to this seemingly apparent contradiction lies in EGCG effectiveness as an innate immune stimulatory by


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mechanisms such as upregulation of phagocytic capacity of phagocytic cells like macrophages and DCs, as well as certain proinflammatory cytokines like TNF␣, crucial for an effective immune response. It is also important to note that upregulation of various parameters often associated with a heightened immune response is not always beneficial to the host in combating microbial infection. Sepsis is a severe and systemic illness caused by the excessive inflammatory response to microbial infections, often with fatal results. Both Gram-negative and Gram-positive bacterial infections are capable of causing sepsis. In this respect, medicinal properties of plant-derived agents need to be considered from the aspect of not only their ability to stimulate immune activity but also their possible role in downregulating excessive inflammatory responses. Ginsan, an acidic polysaccharide prepared from Panax ginseng appears particularly effective in this regard. Mice treated with ginsan before bacterial challenge with Staphylococcus aureus (S. aureus) are reportedly highly protected from sepsis-induced death while still maintaining antibacterial capacity. Interestingly, the phagocytic activity of ginsan-treated macrophages was shown to be considerably enhanced against S. aureus. At the same time, the synthesis of inflammatory cytokines such as IL-12 was significantly downregulated at the early phase of sepsis in the mice treated with ginsan before bacterial challenge (Ahn et al., 2006).

8. Immunomodulatory Effects of Natural Products on TLR Expression and Signal Transduction Toll-like receptors (TLRs) are an essential component for bacterial recognition and initiation of signaling pathways necessary for induction of cytokines, chemokines and co-stimulatory molecule expression in immune cells. Interestingly, there are several reports of inhibition of TLR induction by various medicinal plant-derived products such as ginsan in response to bacterial infection. Expression of TLRs, including TLR2, TLR4 and TLR9, as well as the adaptor molecule MyD88 was considerably reduced in peritoneal macrophages treated with ginsan before a subsequent contact with S. aureus (Ahn et al., 2006). It is believed that host pattern recognition proteins belonging to the TLR family are involved in protective innate immune responses against Lp infection. In particular, TLR2 appears to play a defined role in host resistance, at least in the murine model, though other TLRs are likely involved. In the murine host in which macrophages are permissive for Lp infection, the intracellular growth of the pathogen is enhanced within TLR2–/– macrophages compared to WT mice and TLR4–/– macrophages (Akamine et al., 2005). In vivo growth of Lp is also enhanced in the lungs of TLR2 deficient mice, which results in delayed bacterial clearance (Archer and Roy, 2006). Recent reports also demonstrate that the plant-derived tea catechin EGCG inhibits TLR2 induction by BMDCs caused by bacterial infection or treatment with bacterial products. In particular, EGCG inhibited both Lp and LPS induction

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FIGURE 10.3. EGCG inhibits induced TLR2 on DCs infected with a) L. pneumophila (Lp) or b) stimulated with LPS and treated with various concentrations of EGCG analyzed by flow cytometry. Numbers in quadrants reflect percentages rounded to next greater whole integer. Results shown are 1 of 3 independent experiments with similar results.

of TLR2 (Fig. 10.3). TLR2 is considered a signal transducer for Lp since either viable or killed Lp activate DCs in TLR4-deficient mice, but not in TLR2 knockout A/J mice. A similar inhibition of TLR4 induction was also demonstrated in response to Lp infection (Fig. 10.4).

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FIGURE 10.4. EGCG inhibits induced TLR4 on DCs infected with L. pneumophila (Lp) and treated with various concentrations of EGCg analyzed by flow cytometry. Numbers in quadrants reflect percentages rounded up to next greater whole integer. Results shown are 1 of 3 experiments with similar results.


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A common theme among many plant-derived products having medicinal properties is inhibition of relB, a subunit of the NF␬B pathway. The promoter region of the TLR2 gene contains two NF␬B binding sites which upregulate gene transcription (Musikacharoen et al., 2001). Inhibition of NF␬B also results in lowered DC maturation, confirming an important role for this transcription factor in maturation (Rescigno et al., 1998). It appears likely that inhibition of TLR2 upregulation and maturation by bacterial antigens is related to NF␬B, since EGCG inhibits NF␬B p65 translocation in addition to MAPKs such as MAPKs 1⁄2, p38 and JNK. It is not yet clear whether EGCG inhibits NF␬B directly or indirectly, affecting an upstream component of the TLR4 signaling pathways, which then leads to inhibition of TLR2 expression.

9. Summary and Conclusions Nonspecific stimulation of innate immunity and resistance to acute and chronic infectious diseases has been the mainstay of traditional oriental medicine for centuries. Products from various plant species have beneficial properties against various infectious agents and enhance immune responses, especially innate immunity based on macrophages and dendritic cell activity. Specific immunomodulators, including nontoxic components derived from nonpathogenic microorganisms, plant-derived phytostimulators and even bees wax components can stimulate nonspecifically both cellular and humoral immunity to opportunistic intracellular pathogens like Lp. In particular, green tea–derived polyphenol catechins enhance the ability of macrophages to inhibit Lp growth in vitro. The beneficial effect of the active component of green tea EGCG is considered to be due to stimulatory activity on macrophage production of the immunoregulatory cytokines TNF␣ and IFNy, which have autocrine activating effect on macrophages. EGCG also has pronounced cytokine immunomodulatory effects on mouse BMDCs. In particular, EGCG stimulates TNF␣ production, a cytokine known to be very important for phagocytic activity of innate immune cells and extremely important for antibacterial activity of many plant-derived products. However, the immunomodulatory effects of EGCG on BMDCs are complex and, in general, EGCG has an antiinflammatory effect, inhibiting cytokine induction important for adaptive immunity (e.g., IL-12), various inflammatory chemokines (e.g., RANTES, MIP1), co-stimulatory and MHC molecules (CD40, CD86, CD80, MHCI/II), as well as modulation of TLRs (TLR2, TLR4) expression. This anti-inflammatory effect is characteristic of many medicinal plant-derived products regarded for their potent innate immunostimulatory capacity against bacterial infection and is likely an important property of many of these products in protecting the host from excessive inflammation caused by certain pathogens which can lead to sepsis and death of the host. The mechanism of action for this inhibition is often attributed to its inhibitory effects of important inflammatory transcription factors like NF␬B.

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11 Interaction of Legionella pneumophila with Amoeba Maëlle Molmeret, Marina Santic, and Yousef Abu Kwaik

1. Introduction The first recognized outbreak of pneumonia due to Legionella pneumophila occurred in Philadelphia, during the summer of 1976 among 180 persons attending the 56th annual American Legion Convention. Twenty nine patients died, and the disease became known as Legionnaires’ disease (Fraser et al., 1977). Guinea pigs were infected with postmortem lung tissue from the patients with fatal Legionnaires’ disease, and embryonated yolk sacs were inoculated with spleen homogenates from the infected guinea pigs. In January of 1977, a Gram-negative bacterium was isolated and designated L. pneumophila (McDade et al., 1977). The source of the infection during the Legionnaires’ convention was later found to be the air conditioning system in the hotel. It has been documented that the hallmark of Legionnaires’ disease is the intracellular replication of L. pneumophila in the alveolar spaces. At least 48 species of legionellae have been identified, some of which are associated with disease while others are environmental isolates and whether they can cause disease is not known (Adeleke et al., 2001). L. pneumophila is responsible for more than 80% of cases of Legionnaires’ disease, and among the 13 serogroups of L. pneumophila, serogroup 1 is responsible for more than 95% of cases of Legionnaires’ disease. It is estimated that L. pneumophila is responsible for at least 25,000 cases of pneumonia per year in the US, which is most probably an underestimate due to the difficulty in bacterial isolation from clinical samples. Since L. pneumophila is the most frequent cause of Legionnaires’ disease, most pathogenic and environmental studies have focused on L. pneumophila. Free-living amoebae are important predators controlling microbial communities. They are ubiquitous and have been isolated from various natural sources such as soil, freshwater, salt water, dust, and air. Although their abundance in soil is only limited, they have been implicated in the stimulation of phosphorus and nitrogen turnover and thus play an important role in soil ecosystems (RodriguezZaragoza, 1994). Free-living amoebae are also frequently isolated from anthropogenic ecosystems, such as tap water, air condition units, and cooling towers, feeding on the microbial biofilms present in those systems (Sheehan et al., 2005; 185

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Murga et al., 2001). However, several bacteria have developed mechanisms to survive phagocytosis by free-living amoebae and are able to exploit them as hosts (Molmeret et al., 2005). Transient association with amoebae have been reported for a number of different bacteria including Legionella pneumophila, mycobacterium sp., Francisella tularensis, or Escherichia coli O157, among others (Abu Kwaik, 1996; Bozue and Johnson, 1996; Rowbotham, 1986; Newsome et al., 1985). As most of these bacteria are human pathogens, amoebae have been suggested to represent their environmental reservoirs, acting as “Trojan horses” of the microbial world (Molmeret et al., 2005; Barker and Brown, 1994). To date only the interaction of L. pneumophila, a facultative intracellular pathogen of humans causing Legionnaire’s disease, with free-living amoebae has been studied in greater detail. In 1980, Rowbotham described the ability of L. pneumophila to multiply intracellularly within protozoa (Rowbotham, 1980). Since then, L. pneumophila has been described to multiply in many species of protozoa, and this host–parasite interaction is central to the pathogenesis and ecology of L. pneumophila. L. pneumophila has a very similar intracellular fate within both mammalian and protozoan cells. Intracellular multiplication of Legionella with protozoa such as Acanthamoeba polyphaga and macrophages requires the Dot/Icm secretion system for biogenesis of the phagosome and intracellular replication (Segal and Shuman, 1999; Gao et al., 1997). The dot/icm genes are located in two different regions. The region I includes seven genes (dotA–D,icmV,W,X) and the larger region II contains the remaining 17 members (icmT,S,R,Q,P,O,N,M,L,K,E,G,C,D,J,B,F ) (Christie and Vogel, 2000; Segal and Shuman, 1997; Brand et al., 1994; Berger and Isberg, 1993). The dot/icm genes of the Type IV secretion system are thought to encode proteins involved in the translocation of effector molecules into the host cell that will prevent the bacteria from being killed (Chen et al., 2004; Luo and Isberg, 2004; Conover et al., 2003; Nagai et al., 2002; Christie and Vogel, 2000). The icm/dot loci are highly similar to the transfer region of plasmid R64 and other IncI1 plasmids, which suggests that icm/dot virulence genes share a common ancestor with plasmid conjugation system (Sexton and Vogel, 2002; Komano et al., 2000; Segal et al., 1999; Segal and Shuman, 1999; Segal et al., 1998; Vogel et al., 1998). It is not known whether the icm/dot genes derive from a single plasmid, which has been separated into the two gene clusters, or there were the result of multiple gene transfer events. However, only Coxiella burnettii has homologs of the full icm/dot genes, which are contained in a single locus (Feldman et al., 2005; Seshadri et al., 2003; Zamboni et al., 2003; Zusman et al., 2003). Besides the dot/icm Type IVB secretion system, L. pneumophila possess a second Type IV secretion system, the lvh/lvr genes that surprisingly are not involved in its virulence (Segal et al., 1999). The Type IV secretion system has been shown to be the main virulence system of L. pneumophila.

11. Interaction of Legionella pneumophila with Amoeba

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2. Legionellae: A Facultative Intracellular Pathogen of Free-Living Amoebae Legionella pneumophila is a Gram-negative facultative intracellular bacillus, responsible for Legionnaire’s disease. It replicates within evolutionary-distant eukaryotic host cells such as protozoa and mammalian macrophages (Fig.11.1). In aquatic environments, L. pneumophila is ubiquitous and grows within protozoan hosts. At least 14 species of amoebae and 2 species of ciliated protozoa have been shown to support intracellular replication of L. pneumophila (Shadrach et al., 2005; Fields, 1996). Among the most predominant amoebae in water sources are Hartmannellae and Acanthamoebae, which have been also isolated from water sources associated with Legionnaires’ disease outbreaks (Fields, 1996). Interaction between L. pneumophila and protozoa is considered to be central to the pathogenesis and ecology of L. pneumophila (Molmeret et al., 2005; Harb et al., 2000; Rowbotham, 1986). In humans, L. pneumophila reaches the lungs after inhalation of contaminated aerosol droplets (Fields, 1996; Fliermans, 1996). The main sources of contaminated water droplets are hot water and air conditioning systems, but the bacteria have been isolated from fountains, spas, pools, dental and hospital units, and other man-made water systems (Fliermans, 1996). No person-to-person transmission has ever been described. Once in the lungs, L. pneumophila are ingested by alveolar macrophages, which are thought to be the major site of bacterial replication (Molmeret et al., 2005). This results in an acute and severe pneumonia. Approximately one-half of the 48 species of Legionella have been associated with human disease. L. pneumophila is responsible for 95% of cases of Legionnaires’ disease. However, all the Legionella species under appropriate conditions may be capable of intracellular growth

a

b

U937

Lpn

A. polyphaga

Lpn N

N

2

2

FIGURE 11.1. L. pneumophila replicate in both macrophages and protozoa. Representative Electron microphages of U937 cells (a) and A. polyphaga (b) infected by L. pneumophila at 24 h. Lpn for L. pneumophila and N for Nucleus. Adapted from Molmeret et al. (2004).

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and infliction of human disease. Infections due to less common species of legionellae are not frequently diagnosed and reported, and are less studied than L. pneumophila (Molmeret et al., 2005; Fields et al., 2002). In addition to recognized Legionella species, a number of Legionella-related bacteria-designated Legionella-like amoebal pathogens (LLAPs) (Rowbotham, 1986; Rowbotham, 1983) have been described (Adeleke et al., 1996). Interestingly, many LLAPs have been associated with Legionnaires’ disease (Marrie et al., 2001; Birtles et al., 1996). In contrast to other Legionella species, however, most of the LLAPs cannot be cultured in vitro on artificial media, but are isolated by co-culture with protozoa (Fields, 1996). Considering that approximately 50% of the 0.5 million annual cases of pneumonia in the US are of unknown etiology, the LLAPs may be responsible for at least some of these cases. The recent developments in using the polymerase chain reaction for bacterial identification in environmental samples will facilitate better identification of legionellae and LLAPs. Further cellular and molecular biology studies are needed to better understand the intracellular life of these endosymbionts.

3. The Role of Amoebae in Persistence of Legionella in the Environment It is most likely that the association of legionellae with protozoa is a major factor in continuous presence of the bacteria in the environment. Many strategies have been used to eradicate legionellae from sources of infection in water and plumbing systems that have been associated with disease outbreaks (Molmeret et al., 2005). These strategies include chemical biocides such as chlorine, overheating of the water, and UV irradiation (Biurrun et al., 1999; Kool et al., 1999; Muraca et al., 1987). Such interventions have been successful for short periods of time after which the bacteria reappear in these sources (Biurrun et al., 1999; Yamamoto et al., 1991). Thus, eradication of L. pneumophila from the environmental sources of infection requires continuous treatment of the water with agents such as monochloramine or copper–silver ions in addition to maintenance of the water temperature above ~55°C (Darelid et al., 2002; Kusnetsov et al., 2001; Hoebe and Kool, 2000; Kool et al., 1999). Compared to in vitro-grown L. pneumophila, amoebae-grown bacteria have been shown to be highly resistant to chemical disinfectants and to treatment with biocides (Barker et al., 1992). Amoebae-grown L. pneumophila have been shown to manifest a dramatic increase in their resistance to harsh environmental conditions such as fluctuation in temperature, osmolarity, pH, and exposure to oxidizing agents (Abu Kwaik et al., 1997). Protozoa have been shown to release vesicles containing L. pneumophila that are highly resistant to biocides (Berk et al., 1998). The ability of L. pneumophila to survive within amoebic cysts further contributes to resistance of L. pneumophila to physical and biochemical agents used in bacterial eradication (Barker et al., 1992, 1995). It is likely that eradication of the bacteria from


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the environment should start by preventing protozoan infection, an integral part of the infectious cycle of L. pneumophila. Extracellular L. pneumophila is more susceptible to environmental conditions and is not protected from biocides and disinfectants.

4. The Role of Amoebae in Pathogenesis of Legionella There are many lines of evidence to suggest that protozoa play major roles in transmission of L. pneumophila as infectious particles for Legionnaires’ disease (Molmeret et al., 2005; Rowbotham, 1980). First, many protozoan hosts have been identified that allow intracellular bacterial replication, the only documented means of bacterial amplification in the environment (Harb et al., 2000; Abu Kwaik et al., 1998b; Fields, 1996). Second, in outbreaks of Legionnaires’ disease, amoebae and bacteria have been isolated from the same source of infection and the isolated amoebae support intracellular replication of the bacteria (Fields et al., 1990). Third, following intracellular replication within protozoa, L. pneumophila exhibit a dramatic increase in resistance to harsh conditions including high temperature, acidity, and high osmolarity, which may facilitate bacterial survival in the environment (Abu Kwaik et al., 1997). Fourth, intracellular L. pneumophila within protozoa are more resistant to chemical disinfection and biocides compared to in vitro-grown bacteria (Barker et al., 1992, 1993, 1995). Fifth, protozoa have been shown to release vesicles of respirable size that contain numerous L. pneumophila. The vesicles are resistant to freeze-thawing and sonication, and the bacteria within the vesicles are highly resistant to biocides (Berk et al., 1998). Sixth, following their release from the protozoan host, the bacteria exhibit a dramatic increase in their infectivity for mammalian cells in vitro (Cirillo et al., 1994). In addition, it has been demonstrated that intracellular bacteria within H. vermiformis are dramatically more infectious and are highly lethal in mice (Brieland et al., 1997). Seventh, the number of bacteria isolated from the source of infection of Legionnaires’ disease is usually very low or undetectable, and thus, enhanced infectivity of intracellular bacteria within protozoa may compensate for the low infectious dose (O’Brein and Bhopal, 1993). Eight viable but non-culturable L. pneumophila can be resuscitated by co-culture with protozoa (Steinert et al., 1997). This observation may suggest that failure to isolate the bacteria from environmental sources of infection may be due to this “dormant” phase of the bacteria that cannot be recovered on artificial media. Ninth, there has been no documented case of bacterial transmission between individuals. The only source of transmission is environmental droplets generated from man-made devices such as shower heads, water fountains, whirlpools, and cooling towers of air conditioning systems (Fields, 1996). These findings indicate a rather sophisticated host–parasite interaction and a tremendous adaptation of legionellae to parasitize protozoa. This host–parasite interaction is central to the pathogenesis and ecology of these bacteria.

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5. Entry of L. pneumophila into Protozoa The attachment and entry mechanisms of L. pneumophila into its protozoan hosts and variations in their mechanisms have been reported for both amoeba Hartmanella vermiformis and Acanthamoeba spp. Attachment of L. pneumophila to H. vermiformis is mediated by adherence to a protozoan receptor characterized as a putative galactose/N-acetyl-galactosamine (Gal/GalNAc) lectin (Harb et al., 1998; Venkataraman et al., 1997; Mann et al., 1991). Host protein synthesis by A. polyphaga is not required for invasion by L. pneumophila whereas it is required for invasion of H. vermiformis (Harb et al., 1998). Integrins are heterodimeric protein tyrosine kinase receptors that undergo tyrosine phosphorylation upon engagement to ligands, which subsequently results in recruitment and rearrangements of the cytoskeleton. Interestingly, attachment of L. pneumophila to the Gal/GalNAc of H. vermiformis triggers signal transduction events in H. vermiformis that are manifested in dramatic tyrosine dephosphorylation of the lectin receptor and other proteins (Venkataraman et al., 1997, 1998). Similar observations have been obtained upon infection of H. vermiformis by another species of legionellae, L. micdadei (Abu Kwaik et al., 1998a). Among the L. pneumophila-induced tyrosine dephosphorylated proteins in H. vermiformis are the cytoskeletal proteins paxillin, vinculin, and focal adhesion kinase (Venkataraman et al., 1997, 1998). Tyrosine phosphatases have been shown to disrupt the cytoskeleton in mammalian cells. Thus, the induced tyrosine phosphatase activity in H. vermiformis is probably manifested in disruption of the protozoan cytoskeleton to facilitate entry through a cytoskeleton-independent receptor-mediated endocytosis. Interestingly, in addition to these manipulations of the signal transduction of H. vermiformis by L. pneumophila, bacterial invasion is also associated with specific induction of gene expression in protozoa, and inhibition of this gene expression blocks entry of the bacteria (Abu Kwaik et al., 1994). Following this initial host–parasite interaction, uptake of L. pneumophila by A. castellanii occurs by coiling phagocytosis (Abu Kwaik, 1996; Bozue and Johnson, 1996). However, the uptake of L. pneumophila by H. vermiformis occurs mainly through cup-shaped invaginations (or zipper phagocytosis) on the surface of the amoeba, in addition to occasional coiling phagocytosis (Abu Kwaik, 1996; Bozue and Johnson, 1996). Human macrophages are able to phagocytose heat- or formalin-killed L. pneumophila by coiling phagocytosis (Horwitz, 1984), which indicates that coiling phagocytosis does not play a role in the intracellular fate of L. pneumophila. However, the infectivity of A. castellanii and macrophages by L. pneumophila has been shown to be similar (Hilbi et al., 2001). In addition, the adherence receptors of macrophages used by L. pneumophila does not seem to affect its intracellular survival profoundly as the bacterium multiplies within phagocytes after entry under different opsonizing or nonopsonizing conditions (Cirillo et al., 1999; Marra et al., 1990; Payne and Horwitz, 1987; Horwitz and Silverstein, 1981). Uptake of L. pneumophila by another protozoan host, A. polyphaga, is not completely blocked by Gal or GalNAc and is associated with partial tyrosine dephosphorylation of a 170 kDa


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protein, which may be related to the Gal/GalNAc lectin of H. vermiformis (Harb et al., 1998). Thus, entry of the bacteria into A. polyphaga is partially mediated by the Gal/GalNAc lectin and additional receptors may be involved for bacterial attachment and entry. The heterogeneity in the uptake mechanisms of L. pneumophila into H. vermiformis and A. polyphaga has been confirmed using invasion defective mutants of L. pneumophila. Several mutants that were severely defective in attachment to A. polyphaga exhibited minor reductions in attachment to H. vermiformis (Harb et al., 1998). The mode of entry into mammalian and protozoan host cells have been shown to occur by both dot/icm dependent and independent mechanisms (Bandyopadhyay et al., 2004; Hilbi et al., 2001; Watarai et al., 2001). Studies have shown that phagocytosis of wild-type L. pneumophila is more efficient than uptake of dot/icm mutants within macrophages and A. castallanii, indicating that this mechanism is independent of adherence receptors (Hilbi et al., 2001; Watarai et al., 2001). However, when the hosts are infected with stationary-phase cultures that have been incubated overnight in pH 6.4 buffer, a treatment which enhances the resistance to acid, hydrogen peroxise and antibiotics stress, entry into A. castellanii and macrophages do not require functional dot/icm genes (Bandyopadhyay et al., 2004). In addition, a “repeats in structural toxin” (RTX) gene, rtxA, has also been shown to play a role in adherence and entry and replication within human macrophages, A. castellanii, and in vivo (Cirillo et al., 2000, 2001). These data indicate the remarkable adaptation of L. pneumophila for attachment and invasion into different host cells.

6. Intracellular Trafficking and Multiplication of L. pneumophila Similar to its intracellular fate within macrophages, L. pneumophila is enclosed, after entry into amoebae, in a phagosome surrounded by host cell organelles such as mitochondria, vesicles, and a multilayer membrane derived from the rough endoplasmic reticulum (RER) of amoeba (Tilney et al., 2001; Abu Kwaik, 1996; Swanson and Isberg, 1995; Horwitz, 1983b). The Legionella-containing phagosome (LCP) evades endocytic fusion within mammalian and protozoan cells (Abu Kwaik, 1996; Bozue and Johnson, 1996; Horwitz, 1983a), it intercepts early secretory vesicles exiting the ER, and acquires proteins involved in ER-to-golgi vesicle traffic such as Rab1 and Sec22b (Kagan et al., 2004). Within few minutes of biogenesis, the LCP is remodeled into an ER-derived replicative vacuole (Derre and Isberg, 2004; Kagan et al., 2004; Kagan and Roy, 2002; Tilney et al., 2001; Abu Kwaik, 1996; Horwitz, 1983b). Few hours after internalization, and formation of the ER-derived replicative organelle, bacterial replication is initiated. Both evasion of endocytic fusion and recruitment of early secretory vesicles as they exit the ER are controlled by the Dot/Icm type IV secretion system (Derre and Isberg, 2004; Kagan et al., 2004; Kagan and Roy, 2002; Tilney et al., 2001; Abu Kwaik, 1996; Horwitz, 1983b). Most or all of the

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Dot/Icm structural proteins have been shown to be essential for biogenesis of the LCP and for intracellular multiplication within both protozoa and mammalian cells (Segal et al., 1998; Vogel et al., 1998). The role of the RER in the intracellular infection is unknown, but the RER is not required as a source of proteins for the bacteria (Abu Kwaik, 1998). Interestingly, examination of the intracellular infection of macrophages, alveolar epithelial cells, and protozoa by another legionellae spp., L. micdadei, showed that within all of these host cells, the bacteria were localized to RER-free phagosomes (Gao et al., 1999). Whether other legionellae species replicate within RER-free phagosomes has yet to be determined. Recently, the soil amoeba, Dictyostelium discoideum, has been studied as a new host model for Legionella, in particular to understand how Legionella establish its replicative niche within phagocytic host cells. The advantages of using this model for the infection of Legionella is that D. discoideum can be genetically manipulated, that cellular markers are commercially available, and more importantly, the growth of L. pneumophila is similar to that in macrophages and fresh-water amoebae including the requirement for a functional dot/icm Type IV secretion system (Hagele et al., 2000; Solomon and Isberg, 2000). D. discoideum is found in soil as a unicellular free-living amoeba that feeds on bacteria. Under starvation conditions, the organism undergoes a complex developmental cycle during which it aggregates to form a multicellular motile phototactic slug. This slug can develop into a fruiting body forming viable spores supported by a column of stalk. In a rtoA mutant of D. discoideum where vesicle trafficking event is lowered, the intracellular growth of L. pneumophila is depressed (Li et al., 2005). In addition, Cytoskeleton-associated proteins and calcium-binding proteins of the ER, calnexin and calreticulin, specifically influence the uptake and the intracellular growth of L. pneumophila within D. discoideum (Fajardo et al., 2004). Therefore, ER recruitment plays an important role in the intracellular multiplication of L. pneumophila within this host. One of the hypotheses regarding the role of the recruitment of ER to the LCP is that autophagy mechanism involved in cellular homeostasis was highjacked by Legionella in order to establish its replicative niche. As macroautophagy genes have been identified in D. discoideum, they have been used to show that macroautophagy is dispensable for the intracellular multiplication of L. pneumophila in D. discoideum (Otto et al., 2004). Therefore, it is rather clear that ER-derived vesicles and proteins are part of a system that leads to the establishment of the replicative vacuole of L. pneumophila but autophagy does not appear play a role in this mechanism within Legionella protozoan hosts.

7. Role of the dot/icm Genes in Evasion of the Endocytic Pathway The main virulence system of L. pneumophila is the dot/icm Type IV secretion system. Because the Dot/Icm secretion system is ancestrally related to Type IV secretion systems that mediate conjugal DNA transfer between bacteria (Christie


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and Vogel, 2000), L. pneumophila may utilize this transporter to transfer macromolecules into the host cell to evade endocytic fusion (Roy and Tilney, 2002). The Dot/Icm-mediated transfer is thought to occur through the insertion of a pore in the host cellular membrane through which the effector proteins are transported (Kirby and Isberg, 1998; Kirby et al., 1998). With few exceptions, the function of individual Dot/Icm proteins is unknown. However, the dot/icm genes are present in all tested Legionella species (Alli et al., 2003). Most of the dot/icm genes required for intracellular growth within human cells are also required for intracellular growth in the protozoan host Acanthamoeba castellanii (Segal and Shuman, 1999). Although some loci have been shown to be only essential for the intracellular growth of L. pneumophila in macrophages (Gao et al., 1998), numerous loci have been identified as essential for survival and intracellular replication of L. pneumophila in A. polyphaga or H. vermiformis and in macrophages (Stone et al., 1999; Gao et al., 1997; Cianciotto and Fields, 1992). D. discoideum has been shown to support intracellular multiplication of L. pneumophila (Hagele et al., 2000; Solomon and Isberg, 2000; Solomon et al., 2000). As said above, the intracellular fate of L. pneumophila is very similar in infected D. discoideum to that in macrophages, including the recruitment of RER, evasion of lysosomal fusion (Solomon et al., 2000), and dependence of intracellular growth on dot/icm gene functions (Solomon et al., 2000). The similarity between the infection by L. pneumophila of different protozoa supports the idea that the ability of L. pneumophila to parasitize macrophages and hence to cause human disease is a consequence of its prior adaptation to intracellular growth within protozoa. The Type IV secretion system of L. pneumophila is though to be an effective apparatus to translocate effector molecules into the host cell and modulate the host cell physiology in order for the bacteria to establish a replicative niche by recruiting ER-derived vesicles and evading the endocytic pathway. Genetic screens that enable the identification of the set of 26 dot/icm genes have failed to identify the genes encoding the Type IV secretion system substrates secreted into host cells (Vogel et al., 1998; Segal and Shuman, 1997; Berger and Isberg, 1993). Different strategies have been used for this purpose. First, strategies which are not based on intracellular growth defect of the bacteria but based on homology to eukaryotic protein have allowed the identification of Type IV secretion system effectors such as RalF, LepA, and LepB (Chen et al., 2004; Nagai et al., 2002). Most of these substrates do not express an intracellular replication-deficient phenotype (or a minor one) within both mammalian and protozoan host cells, which explain why they had not been isolated previously in genetic screens for intracellular growth mutants. RalF protein, containing a Sec7-homology domain, is produced by L. pneumophila and is injected into the host cells by the Dot/Icm transporter, functioning as an exchange factor that activates members of the ARF protein family (Nagai et al., 2002, 2005). LepA and LepB, which have a weak homology to SNAREs have been shown to be delivered to host cells by a type IV secretion system-dependent mechanism. The double mutant of the paralogs lepA lepB exhibited a defect in release of L. pneumophila-containing “fecal” or

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“respirable” vesicles from A. castellanii and D. discoideum (Chen et al., 2004). Second, a strategy based on the fact that some translocated proteins also function to maintain the integrity of the Dot/Icm translocator and mutations that destroy this function are predicted to result in a Dot/Icm complex that poisons the bacterium, resulting in reduced viability, has allowed to identify an effector called LidA. This substrate LidA, identified by a complex genetic screen, has been shown to be associated with the cytoplasmic face of the L. pneumophilacontaining phagosome (Conover et al., 2003). Third, an interbacterial protein transfer assay is an another strategy used to identify the substrate SidC (substrate of icm/dot complex) identified among other Sid substrates also secreted into macrophages (Luo and Isberg, 2004). Finally, some substrates have been identified using chaperone proteins belonging to the dot/icm genes secretion system, as bait. IcmS has been hypothesized to serve as a chaperone for secreted substrates since it is predicted to be located in the bacterial cytoplasm, has no homology to bacterial conjugal transfer proteins, and has similar biochemical properties of secretion chaperones from the type II secretion system (Feldman and Cornelis, 2003; Coers et al., 2000). IcmS has been used as a bait to identify potential secreted substrates such as SidE family proteins (SdeA, SdeB, SdeC, SidE) (Bardill et al., 2005). SidE family proteins have been shown to be secreted by L. pneumophila at early stages of infection of macrophages across the phagosomal membrane and have been shown to be required for full virulence in Acanthamoeba castellanii although individual deletions of a number of substrates had a modest or no effect in intracellular replication (Luo and Isberg, 2004) probably resulting from the export of functionally redundant proteins by the Type IV secretion system. In addition, as the complex IcmS–IcmW forms a stable protein complex and plays an important role in substrate translocation, IcmW has also been used as a bait in a yeast two-hybrid system to identify substrate proteins translocated into host cells by the type IV secretion system (Ninio et al., 2005). IcmW-interacting proteins (Wips) have been recovered, including SidG and SidH identified previously with the interbacterial protein transfer screen (Luo and Isberg, 2004) as well as WipA, which is translocated into mammalian host cells. A paralog called wipB, which is also translocated into macrophages by the Type IV secretion system, was found in the Legionella genome data base and the double mutant wipA wipB had no growth defect in A. castallanii (Ninio et al., 2005). The functions of the identified substrates are under investigation. There are probably hundred of effectors that need to be identified in order to understand how Legionella establish a successful replicative niche within its host cells. Some of these substrates may also be host-dependent and be functional only in one of the various host cells of Legionella.

8. Egress of L. pneumophila from Amoebae A detailed ultrastructural analysis of late stages of intracellular replication has been performed to examine egress of L. pneumophila from both macrophages and amoebae by electron microscopy (Molmeret et al., 2004). The membrane of the


a

195

c

N N

2

2 N

b

d

Lp

Lp

Lp

Lp

V

Lp

M Lpn

V V Lp Lp

500

V 500 nm

FIGURE 11.2. L. pneumophila are contained within intact phagosome at 8 h post-infection and are cytoplasmic at 24 h post-infection. Representative electron micrographs of L. pneumophila-infected macrophages 8 h post-infection are shown in (a) and (b). The membrane of the LCP is intact as indicated by the thin arrows. Lpn for L. pneumophila, N for Nucleus. Representative electron micrographs of L. pneumophila-infected macrophages at 24 h post-infection are shown in (c) and (d). L. pneumophila (Lpn) are cytoplasmic where bacteria are surrounded by numerous vesicles (V), lysosomal contents (white arrows), mitochondria (M), and amorphous elements (A). No distinguishable phagosomal membrane surrounds the bacteria. N for nucleus. Adapted from Molmeret et

LCP within both macrophages and Acanthamoeba polyphaga is intact up to 8 h post-infection (Molmeret et al., 2004). However, at 12 h, the majority of the LCPs are disrupted within both hosts, while the plasma membrane remains intact (Molmeret et al., 2004). At 18 and 24 h post-infection, cytoplasmic elements such as mitochondria, lysosomes, vesicles, and amorphous material are dispersed among the bacteria and these bacteria are considered cytoplasmic (Molmeret et al., 2004). Thus, by 18 h–24 h post-infection, the majority of the remaining host

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cells harbor cytoplasmic bacteria and this transient cytoplasmic presence of L. pneumophila precedes lysis of the plasma membrane. Interestingly, within both macrophages and amoebae, bacterial replication proceeds in the cytoplasm. Therefore, the phagosomal membrane is disrupted first, rather than by simultaneous lysis of both the phagosomal and the plasma membranes. These disruptions of LCP may be the result of multifactorial events linked to apoptosis, the pore-forming activity (PFA) of the Type IV secretion system and mechanical pressure due to the increase of the phagosomal size (Kirby et al., 1998).

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Index

Acanthamoeba castellani, 12–44 Acetyl-phosphate, 101–102 Adaptive immune response and Legionella immunopathogenesis, 156–157 Agrobacterium tumefaciens Vir system, 13 AhpC, see Alkylhydroperoxide reductase enzymes AhpCAhpD complex, 125 Alkylhydroperoxide reductase enzymes, 125 Amino acid transporter (SLC1A5), 114 Aminoglycoside phosphotransferase (3’)-IIIa (APH), 21 Amoeba proteus, 66 Amoebae-grown L. pneumophila, 188 Ankyrin repeat proteins, 9 Anti-sigma factors FlgM and factor B, 16 Anti-TNFα antibody treatment and L. pneumophila infection, 155 Antibacterial activity and L. pneumophila, 155 Antigen presenting cells, 157 APCs, see Antigen presenting cells Bacterial Ccm systems, 39 BCYEα medium, 123 BipA, virulence regulator, 11 BMDCs, see Bone marrow derived dendritic cells BMMs, see Bone marrow derived macrophages Bone marrow derived dendritic cells, 176 Bone marrow derived macrophages, 176 Bordetella pertussis, 49 Broth model, 97–98 Burkholderia pseudomallei, 10 Burkholderia thailandensis, 10 C. burnetii, 9 Capsaicin (CAP), 176 Carbon storage regulatory (Csr) system, 105–106

CAS assay, 34 CDM, see Chemically defined medium Cell mediated immune, 151–152 Chemically defined medium, 34 Chemokines, 156 Chemokines from inflammatory cells4,21,31,32,34,50, 154 Chlamydia pneumoniae, pathogen, 10 CMI, see Cell mediated immune competence- and adherence-associated pili (CAP), 68 CpxA/CpxR two-component systems, 54 Cre/loxP-based system, 13 CsrA repression of transmissive traits, 96 CuZn enzyme, 125 Cyclosporine immunosuppressive drug, 158 Cytochrome c maturation (ccm) locus mutations, 38–39 Cytochrome c reductase PetA, 41 Cytokines modulation by natural products, 175–176 Cytoplasmic ATPase, 40 Defective organelle trafficking system, 13 Delayed type hypersensitivity (DTH) reactions, 156–157 Delta4 expression, 161–162 Dendritic cells (DCs), 176 Dictyostelium discoideum, 68, 192 Direct fluorescent antibody test, 140 dot system, see Defective organelle trafficking system dot/icm genes discovery, 50 Dot/Icm type-IV secretion system, 13–14, 192–193 adaptor/chaperone complexes, 53–54 effector export regulation of, 54 genetic regulation of, 54–55

203

204

Index

LCV biogenesis, 58–59 proteins effector and functions, 55–58 regulation of, 107 structural components of, 52–53 types of, 49 DotH/DotG/DotF subcomplex, 53 dotU/icmF mutant, 53 E3-Ubiquitin ligase, 9 ECF factors, see Extracytoplasmic sigma factors ECF-type sigma factor rpoE, 16 EGCG inhibition induced TLR4 on DCs infected with L. pneumophila, 180 Endocytic pathway, dot/icm genes role in evasion, 192–194 Endoplasmic reticulum-Golgi intermediate compartment, 56 Entamoeba histolytica, 10 Epigallocatechin gallate, 175 Epigallocatechin gallate, antibacterial and antiviral effects, 174–175 ERGIC, see Endoplasmic reticulum-Golgi intermediate compartment Erythrobacter litoralis, 10 Escherichia coli autotransporters AIDA-I and Ag43, 12 K12 amoebae, 10 espC pathogenicity island, 12 EspC toxin, 12 Eukaryotic signal transduction pathways, 10 Eukaryotic-like domains in genome of pathogens, 11 Extracytoplasmic sigma factors, 125 Extrapulmonary manifestations of LD, 139 F-box proteins, 9 feoB gene in intracellular infection, 37 Ferric-regulated gene A (frgA), 35 Flagella expression, 12 Flagella-mediated cell motility, 12 FliA sigma factor, 103 Fluoroquinolone administration, 142 Free-living amoebae, 185 Fur, transcriptional repressor, 33–34 Gal/GalNAc lectin, see Galactose/ N-acetylgalactosamine-inhibitable amoebal lectin Galactose/N-acetylgalactosamine-inhibitable amoebal lectin, 67 GATA-3 knockout mice, 161 GCAT, see Glycerophospholipid:cholesterol acyltransferase GCN5-related N-acetyltransferase, 22

GenBank database, 23 GGDEF-EAL family of regulators, 16, 108 Glycerophospholipid:cholesterol acyltransferase, 42 GNAT, see GCN5-related N-acetyltransferase GTPase ADP-ribosylation factor (Arf)-1, 55 Hartmannella vermiformis amoebae, 36 Heat shock proteins, 126 HeLa cells, growth cycle of L. pneumophila in, 73–74 Helicobacter pylori, 49 Hfq and Small RNAs (sRNAs), 106 Host-cell trafficking pathways, 10 HSPs, see Heat shock proteins icm system, see Intracellular multiplication system icmQ and icmR pore-forming protein, 23 IHF, see Integration host factor IL-4 mRNA upregulationin ex vivo splenocyte cultures, 160 Inner (trans) membrane protein, 40 Inner membrane platform (T2S L, M), 40 Integration host factor, 127 Interleukins IL-1, 152–153 Intracellular multiplication system, 13 iraA and iraB gene, 38 Jagged1 expression, 161 lbtA and lbtB genes, 35 lbtA for legiobactin gene A, 35 LCV, see Legionella-containing vacuole LEE, see Locus of enterocyte effacement Legiobactin discovery of, 34–35 genetics of production, 35–36 lbtA and frgA role in infection, 36 Legionella ferric reductases, 39 Legionella pneumonia, 138 Legionella pneumophila genome amino acid uptake and degradation systems in, 14–15, 98 amoebae role in pathogenesis, 187, 189 amoebae role in persistance of environment, 188 and mortality in IL-4 competent/deficient BALB/c mice, 160 attachment and entry mechanisms protozoa, 190–191 autophagy machinery of, 10 autotransporters of, 12 biofilms association of, 90–91

Index biosynthetic capacity and amino acid auxotrophies, 123–124 bipA gene in, 11 circular genome map of, 3 developmental cycle of, 72–73 distribution and concentration in environment, 85–86 dot/icm mutants, 24 Dot/Icm proteins characteristics of, 51 egress examination of, 194–195 extracellular growth cycle and development, 73–76 features of, 1–2 ferrous iron transport in, 37–38 gene family expansion in, 24–25 gene level variability, 22–23 genetic organization of strains of, 20 growth cycles of, 70–72 host pathogen interaction in, 3–4 immune mechanisms in, 157–164 immunogens of, 152–153 innate immune responses and infection, 153–156 interaction with other organisms, 90 intracellular differentiation, 76–77 iraAB (iron acquisition/assimilation) locus, 38 iron assimilation in, 33–34 Krebs cycle, 121–122 life cycle of, 66–67, 95–96 macrophages of, 153 man made environments of, 88–89 metabolic pathways of, 120 MIF role in virulence of, 77–78 model for regulation of differentiation, 97 morphological events in, 68–69 natural environments of, 87–88 oxidative stress and, 124–125 plasmids of, 20–22 proteins-encoding domains, 7–8 replication in macrophages and protozoa, 187 respiratory form of metabolism, 119 secretion system in, 13–14 strains comparison of, 17–18 synteny plot of chromosomes, 4 transcriptional control of differentiation and sigma factors, 102–103 type II secretion discovery in, 41 U-box and F-box motifs of, 9 vegetative and cyst forms of, 115 Legionella pneumophila Philadelphia, features, 2 Legionella pneumophila strains, protein distribution, 5–6 Legionella virulence gene A (lvgA), 53 Legionella-containing vacuole, 51

205

Legionella-infected dendritic cells EGCG effects on co-stimulatory molecule expression, 177–178 cytokine production, 176–177 chemokine production, 178–179 Legionella-phagosome factor, 55 Legionella-related bacteria-designated Legionella-like amoebal pathogens (LLAPs), 188 Legionella-related endosymbionts, 66 Legionella-stimulated IL-12 production, 162 Legionnaires’ Disease (LD) clinical features of, 137–140 diagnosis of, 140–141 epidemiology of, 133–136 nosocomial cases of, 143–144 outbreaks of, 136–137 treatment of, 141–142 Lens strain circular map of plasmid, 22 Lens strains transcriptional regulators, 16 LepA and LepB gene, 57 Let-intermediate (leti) phase, 105 LetA/LetS two-component system, 96, 104–105 LidA and SidM gene, 56–57 LidA over-expression, 56 lipA (LipA) and lipB (LipB) genes (proteins), 42 Listeria monocytogenes, pathogen, 10 Locus of enterocyte effacement, 12 Lp-resistant BALB/c mice, 177 LpPI-1 pathogenicity island, 19 LpPI-1 tra gene homologs, 19 LPS biosynthesis locus, 116 lsp type II secretion system, 13–14 lss type I secretion system, 14 lvh type-IV secretion system, 18 Lymphocytes and L. pneumophila infection, 158 Lymphoid chemokines, 178 Lysophospholipase A, 42 Lysosomal-associated membrane protein (LAMP)-1, 50 LysR-type and phage-like repressors, 16 Macrophage cultures and L. pneumophila infection, 159 Macrophage infectivity potentiator (MIP) protein, 117 magA gene, Philadelphia 1 specific, 18 Major (T2S G) and minor (T2S H, I, J, K) pseudopilins, 40 Major facilitator (MFP) genes, 114 Major facilitator superfamily of transporters, 99 Major outer membrane protein, 153 map (Map) genes (proteins), 42

206

Index

Mature intracellular form, 76 Met tRNA gene, 19 MHC class I pathway, 177 Mice experimental infection with L. pneumophila, 158 MIF, see Mature intracellular form MOMP, see Major outer membrane protein Monocyte chemotactic protein 3 (CCL7) gene expression, 156 Mouse typhoid disease model, 12 msrA and msrB encoding antioxidant repair enzymes, 18 MviN virulence factor, 12 Mycobacterium tuberculosis, pathogen, 10 Mycoplasma genitalium, 18 MyD88 deficient mice, 162 Neisseria gonorrhea, 18 NOTCH 1 signaling pathway in Th2 cells, 161 OmpS, see Outer membrane protein serogroups Outer membrane protein serogroups, 116–117 Outer membrane secretin, 40 Paris strain circular map of plasmid, 22 Paris strains transcriptional regulators, 16 Peptidyl-prolyl-cis/trans isomerase activity, 117 Phagosomal transporters (Phts) family, 98–100 Phagosome-lysosome fusion, 10, 24 PHBA, see Poly-β-hydroxybutyrate Philadelphia 1 strains transcriptional regulators, 16 Philadelphia outbreak fever, 138 Philadelphia-1strain, lspFGHIJK locus, 41 Phosphatidylinositol(4) phosphate [PI(4)P], 56 PilB and PilC proteins, 41 pilBCD operon, 41 pilD::lacZ fusion strain, 44 plaA (PlaA) genes (proteins), 42 plaC (PlaC) genes (proteins), 42 Plasmid RSF1010, 55 plcA (PlcA) genes (proteins), 42 Pneumococcal pneumonia, 139 Poly-β-hydroxybutyrate, 114 Pontiac fever, 133, 143 ppGpp synthase RelA (icmP), 54 Pre-pseudopilin peptidase (PilD), 40 Pre-pseudopilin peptidase T2S O (PilD), 41 Pre-pseudopilin peptidase/methyltransferase (T2S O), 40 proA/msp (ProA/Msp) zinc metalloprotease, 42 Protozoa and growth of Legionella, 3–4 Pseudomonas aeruginosa, pathogen, 10

Putative galactose/N-acetyl-galactosamine (Gal/GalNAc) lectin, 189 Putative inner membrane efflux protein, 12 Putative virulence factors from sequence analysis, 11–12 RalF gene, 55 Renal manifestations in LD, 139 Repeats in structural toxin (RTX) gene, 191 RGD interaction motif, 12 Rickettsia felis, 9 RNA polymerase (RNAP) core enzyme, 100 RpoN and FleQ sigma factor, 103 RpoN modulator, 16 RpoS stationary phase sigma factor, 54, 103 Saccharomyces cerevisiae, 50 Salmonella typhimurium amoebae, 10 Secretin interactions (T2S C), 40 Sepsis illness, 179 Serine-threonine protein kinases, 10 Serine/threonine protein kinases, 9–10 Serum IL-12 p40/p70 level in L. pneumophila infection, 159 Shield flexneri, pathogen, 10 Sid protein family, 56 SidA-G (substrate of Icm/Dot transporter), 13 Sigma factor-dependent enhancers (FleQ, FleR, PilR), 16 SNAREs, see Synaptosomalassociated protein receptors Sphingomyelin degradation pathway, 9–10 Sphingosine kinase, 9 Sphingosine-1-phosphate lyase, 9 Staphylococcus aureus, 179 STPKs, see Serine/threonine protein kinases Stringent response, 100–101 Symbiobacterium thermophilum, 10 Synaptosomalassociated protein receptors, 56 T helper cells phenotypes of, 158 polarization by chemokines, 163 T-lymphocyte function, 158 T2S D, see Outer membrane secretin T2S E, see Cytoplasmic ATPase T2S F, see Inner (trans)membrane protein T2S FGHIJK proteins, 41 T2S-dependent ProA/Msp metalloprotease, 42 T2S-specific mutants, 41 T4SS receptor for cytoplasmic substrates, 52 Tartrate-sensitive and tartrate-resistant acid phosphatases, 42 TAT secretion system, 14

Index tatAB operon, 14 tatC gene, 14 Tetrahymena vorax, 70 Th1 and Th2 helper cell activity, 157 Thr tRNA gene, 19 TLR plus inhibitor ODN 2088, IL-12 production in DCs infected L. pneumophila, 163 TLR4, see Toll like receptor 4 Toll like receptor 4, 116 Toll-like receptors (TLRs), expression and transduction immunomodulatory effects of natural products, 179–181 Tra proteins, 21 Tumor necrosis factor (TNFα), 152–153 Twin arginine translocation (TAT) pathway, 14 Type II protein secretion (T2S) in gram-negative bacteria, 40 in Legionella pneumophila, 41 role in environmental survival, 44–45 role in pathogenesis, 43–44 Type III secretion system, 12

207

Type IV secretion system of L. pneumophila, 193 Type-IVB secretion system, 13, 24 Type-V secretion system, 14 U-box protein, 9 Vacuole protein sorting (VPS) pathway in S. cerevisiae, 58 Vacuole protein sorting inhibitor proteins, 58 Viable but nonculturable state, 86 Vibrio cholerae, 17 Vips, see Vacuole protein sorting inhibitor proteins VNBC, see Viable but nonculturable state Walker A box motif, 52 Wolbachia pipitentis, 9 Wolinella succinogenes, 17 Ylf and Vips gene, 57–58

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Rapid Detection of Infectious Agents (Infectious Agents and Pathogenesis)

Infectious Diseases and Substance Abuse (Infectious Agents and Pathogenesis)

Mims' Pathogenesis of Infectious Disease

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Legionella Pneumophila: Pathogenesis and Immunity (Infectious Agents and Pathogenesis)

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Opportunistic Intracellular Bacteria and Immunity (Infectious Agents and Pathogenesis)

Opportunistic Intracellular Bacteria and Immunity (Infectious Agents and Pathogenesis)

Microorganisms and Bioterrorism (Infectious Agents and Pathogenesis)

Viral Pathogenesis and Immunity

Staphylococcus Aureus : Infection and Disease (Infectious Agents and Pathogenesis) (Infectious Agents and Pathogenesis)

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