Molecular Paradigms of Infectious Disease: A Bacterial Perspective (Emerging Infectious Diseases of the 21st Century)

Molecular Paradigms of Infectious Disease A Bacterial Perspective

Emerging Infectious Diseases of the 21st Century Series Editor: I.W. Fong Professor of Medicine, University of Toronto Head of Infectious Diseases, St. Michael’s Hospital Recent volumes in this series: MALARIA: GENETIC AND EVOLUTIONARY ASPECTS Edited by Krishna R. Dronamraju and Paolo Arese

INFECTIONS AND THE CARDIOVASCULAR SYSTEM: New Perspectives Edited by I.W. Fong

REEMERGENCE OF ESTABLISHED PATHOGENS IN THE 21st CENTURY Edited by I.W. Fong and Karl Drlica

BIOTERRORISM AND INFECTIOUS AGENTS: A New Dilemma for the 21st Century Edited by I.W. Fong and Ken Alibek

MOLECULAR PARADIGMS OF INFECTIOUS DISEASE: A Bacterial Perspective Edited by Cheryl A. Nickerson and Michael J. Schurr

Cheryl A. Nickerson Michael J. Schurr Editors

Molecular Paradigms of Infectious Disease A Bacterial Perspective

Cheryl A. Nickerson School of Life Sciences Center for Infectious Diseases and Vaccinology The Biodesign Institute Arizona State University Tempe, AZ 85287 USA Cheryl.Nickerson@ asu.edu

Michael J. Schurr Program in Molecular Pathogenesis and Immunity Department of Microbiology and Immunology Tulane University Health Sciences Center New Orleans, LA 70112 USA mschurr @ tulane.edu

Library of Congress Control Number: 2005938810 ISBN-10: 0-387-30917-9 ISBN-13: 978-0387-30917-0

e-ISBN 0-387-32901-3

Printed on acid-free paper.

© 2006 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 in the United States of America. 9 8 7 6 5 4 3 2 1 springer.com

(SPI/EB)

This book is dedicated to the editors’ respective parents, Dr. and Mrs. Max A. Nickerson, and Mr. and Mrs. John C. Schurr, and to Jill Schurr.

Preface

Infectious diseases caused by bacterial pathogens are a leading cause of human death and illness worldwide. In addition to causing significant morbidity and mortality bacterial diseases impose an enormous financial burden on society. Recently, the challenges of effectively treating and preventing bacterial infections have been complicated by (1) the emergence of new organisms and diseases; (2) reemerging strains whose incidence had previously declined; (3) changing patterns of well-known diseases; (4) increased antibiotic resistance in many strains; and (5) the potential misuse of bacteria as agents of bioterrorism. The complexity of these challenges is made even clearer as researchers continue to discover novel mechanisms of bacterial pathogenesis and begin to fully appreciate the diverse ways that bacteria cause disease in humans. Collectively, these issues continue to provide important challenges at both the basic research and clinical levels, and highlight the continued need for an improved understanding of the mechanisms of bacterial pathogenesis. The study of bacterial pathogenesis has changed dramatically over the last decade, as a result of revolutionary changes in biotechnology and our understanding of molecular and cellular biological systems. This information has greatly enhanced our understanding of bacterial pathogens and how they cause disease. Indeed, in light of the genomic era that has dawned since 1995, it has become increasingly apparent that many bacteria utilize similar methods to become successful pathogens. Therefore, this book is structured to emphasize paradigms of infectious disease that have emerged in the last 10 years. This book is designed to provide students (both undergraduates and graduates) of the biological and medical sciences with a fundamental understanding of the complex cellular and molecular processes that are important for bacterial virulence and the infectious disease process. In addition, this book serves as a useful text/reference for scientists and researchers of bacterial pathogenesis. Every chapter starts with a boxed section that provides students with a historical overview of critical discoveries that have been accomplished in that specific area of bacterial pathogenesis. A summary

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Preface

section at the end of each chapter provides a review of the major points of the chapter text. In addition, a question-and-answer section is included at the end of each chapter to help students assess their fundamental understanding of the topic covered. We have arranged the book in three basic parts. The first highlights key techniques and methodologies that have driven recent discoveries in bacterial pathogenesis including basic genetic and molecular techniques, genomics, and genetic analyses that have been used to identify virulence factors. The second focuses on major structures and mechanisms in bacteria that are important for the pathogenesis/virulence of these organisms. The third concentrates on the regulation of these virulence determinants by global regulators. Since interruption of global regulatory mechanisms abrogates many different bacterial virulence determinants, a better understanding of these pathogenic mechanisms will allow the development of novel therapeutics targeted against a wide range of bacterial pathogens. In closing, we would like to thank our respective postdoctoral mentors and role models in bacterial pathogenesis, Dr. Roy Curtiss III (C. Nickerson) and Dr. Vojo Deretic (M. Schurr) for their training, support, and generosity. We would also like to thank our respective families and friends for their patience, support, and understanding during the long process of preparation and production of this book. We are especially grateful to Jamie Dominique for her expert assistance in the formatting of this book and her patience in incorporating our many changes into the manuscript.

Contributors

Gregory G. Anderson, Washington University School of Medicine, Department of Molecular Microbiology, St. Louis, MO 63110 Steven R. Blanke, Department of Microbiology and Molecular Genetics, University of Illinois, Urbana, IL 61801 Chasity Baker, Department of Molecular and Cellular Biology, Tulane University Medical School, New Orleans, LA 70112 Robert T. Cartee, Department of Microbiology, University of Alabama at Birmingham, Birmingham, AL 35294 Lucy Cárdenas-Freytag, Program in Molecular Pathogenesis and Immunity, Department of Microbiology and Immunology, SL38, Tulane University Medical School, New Orleans, LA 70112 Clint Coleman, Program in Molecular Pathogenesis and Immunity, Department of Microbiology and Immunology, Tulane University Medical School, New Orleans, LA 70112 Anders Frisk, Program in Molecular Pathogenesis and Immunity, Department of Microbiology and Immunology, Tulane University Health Sciences Center, New Orleans, LA 70112 Audrey Glynn, Program in Molecular Pathogenesis and Immunity, Department of Microbiology and Immunology, SL38, Tulane University Medical School, New Orleans, LA 70112 Joanna B. Goldberg, Department of Microbiology, University of Virginia Health System, Charlottesville, VA 22908 Barry S. Goldman, Agilix Corporation, New Haven, CT 06519 Conrad Halling, Agilix Corporation, New Haven, CT 06519 Daniel J. Hassett, Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati College of Medicine, Cincinnati, OH 45267-0524 ix

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Contributors

Michael Hensel, Institut für Klinische Mikrobiologie, Immunologie und Hygiene, FAU Erlangen-Nürnberg, D-91054 Erlangen Scott J. Hultgren, Washington University School of Medicine, Department of Molecular Microbiology, St. Louis, MO 63110 James A. Imlay, Department of Microbiology, University of Illinois at Urbana-Champaign, Urbana, IL 61801 Yvonne M. Lee, Washington University School of Medicine, Department of Molecular Microbiology, St. Louis, MO 63110 Lisa A. Morici, Program in Molecular Pathogenesis and Immunity, Department of Microbiology and Immunology, Tulane University Health Sciences Center, New Orleans, LA 70112 Cheryl A. Nickerson, School of Life Sciences, Center for Infectious Diseases and Vaccinology, The Biodesign Institute, Arizona State University, Tempe, AZ 85287 Matthew R. Parsek, Department of Microbiology, Carver College of Medicine, The University of Iowa, Iowa City, IA 52242 Michael J. Schurr, Program in Molecular Pathogenesis and Immunity, Department of Microbiology and Immunology, Tulane University Health Sciences Center, New Orleans, LA 70112 Joshua D. Shrout, Department of Microbiology, Carver College of Medicine, The University of Iowa, Iowa City, IA 52242 James M. Slauch, Department of Microbiology, University of Illinois, Urbana, IL 61801 Craig L. Smith, Washington University School of Medicine, Department of Molecular Microbiology, St. Louis, MO 63110 Mark Soboleski, Program in Molecular Pathogenesis and Immunity, Department of Microbiology and Immunology, SL38, Tulane University Medical School, 1430 Tulane Avenue, New Orleans, LA 70112 James W. Wilson, School of Life Sciences, Center for Infectious Diseases and Vaccinology, The Biodesign Institute, Arizona State University, Tempe, AZ 85287 Jennifer K. Wolf, Department of Microbiology, University of Virginia Health System, Charlottesville, VA 22908 Dan Ye, Department of Microbiology and Molecular Genetics, University of Illinois, Urbana, IL 61801 Janet Yother, Department of Microbiology, University of Alabama at Birmingham, Birmingham, AL 35294 Daoguo Zhou, Department of Biological Sciences, Purdue University, West Lafayette, IN 47907

Contents

Preface Contributors Chapter 1: Chapter 2:

Chapter 3:

Chapter 4: Chapter 5: Chapter 6: Chapter 7:

Chapter 8: Chapter 9: Chapter 10: Chapter 11:

vii ix Genetic Analysis of Bacterial Pathogenesis James M. Slauch Genetic Exchange in Bacteria and the Modular Structure of Mobile DNA Elements James W. Wilson Genomics and the Use of Genomic Tools to Study Pathogenic Bacteria Barry S. Goldman and Conrad Halling Pathogenicity Islands and Bacterial Virulence Michael Hensel Capsules Robert T. Cartee and Janet Yother Bacterial Cell Walls Jennifer K. Wolf and Joanna B. Goldberg Mechanisms of Bacterial Adhesion and Consequences of Attachment Gregory G. Anderson, Yvonne M. Lee, Craig L. Smith, and Scott J. Hultgren Bacterial Invasion into Non-Phagocytic Cells Daoguo Zhou Bacterial Protein Secretion Mechanisms James W. Wilson Toxins as Host Cell Modulators Dan Ye and Steven R. Blanke Quorum Sensing: Coordinating Group Behavior Through Intercellular Signals Joshua D. Shrout and Matthew R. Parsek

1 34

78

115 138 176 207

247 274 321 404

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Contents

Chapter 12:

Chapter 13:

Chapter 14:

Chapter 15:

Index

The Role of Sigma Factors in Regulating Bacterial Stress Responses and Pathogenesis Clint Coleman, Chasity Baker, and Cheryl A. Nickerson Two-Component Regulatory Systems Lisa A. Morici, Anders Frisk, and Michael J. Schurr Oxidative Stress Systems in Bacteria: Four Model Organisms Daniel J. Hassett and James A. Imlay Bacterial Biowarfare Agents Mark Soboleski, Audrey Glynn, and Lucy Cárdenas-Freytag

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Chapter 1 Genetic Analysis of Bacterial Pathogenesis JAMES M. SLAUCH

1. 2.

3.

4. 5.

Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Fusion-based Techniques for Identification of Virulence Genes . . . . 3 2.1. TnPhoA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 2.2. In vivo Expression Technology . . . . . . . . . . . . . . . . . . 5 2.3. Variations on the IVET Theme . . . . . . . . . . . . . . . . . 13 Transposon-based Techniques for Identification of Virulence Genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 3.1. Signature-tagged Mutagenesis . . . . . . . . . . . . . . . . . . 16 3.2. Genomic Analysis and Mapping by in vitro Transposition. . . 17 3.3. Transposon Site Hybridization. . . . . . . . . . . . . . . . . . 19 Classic Bacterial Genetics in an Animal Model . . . . . . . . . . . . 22 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

Historical Landmarks 1975

1976

So, Boyer, Betlach, and Falkow clone the gene encoding the heat stabile enterotoxin (ST) from enterotoxigenic Escherichia coli, the first example of cloning a “virulence factor” (So et al., 1976). Casadaban develops a generally applicable technique for constructing transcriptional fusions to the lac operon (Casadaban, 1976). These techniques become more facile over the next decade providing powerful methods to study transcriptional and post-transcriptional regulation in bacteria (see Silhavy and Beckwith, 1985; Silhavy et al., 1984).

Department of Microbiology & College of Medicine, University of Illinois at Urbana/Champaign, Urbana, IL 61801

1

2

1981

1985

1985

1987 1987

1988 1993

1995

1995 1998

2001

James M. Slauch

Garfinkel and Nester use random transposon Tn5 mutagenesis and screen for avirulent Agrobacterium tumefaciens mutants (Garfinkel and Nester, 1980). This is quickly followed by a series of studies where the investigators used transposon mutagenesis and screened for mutations that affect virulence or symbiosis in animals and plants (e.g., Chakravorty et al., 1982; Corbin et al., 1982; Meade et al., 1982; Nida and Cleary, 1983; Normark et al., 1983; Portnoy et al., 1983; Waalwijk and de Graaff, 1983; Weiss et al., 1983; Zimmerman et al., 1983). Isberg and Falkow identify Yersinia Invasin by random cloning into non-invasive E. coli and selecting for invasive isolates (Isberg and Falkow, 1985). Manoil and Beckwith and Hoffman and Wright develop PhoA translational fusions to identify envelope proteins in Gram-negative bacteria (Manoil and Beckwith, 1985; Hoffman and Wright, 1985). Taylor, Miller, Furlong, and Mekalanos use TnPhoA to identify the toxin coregulated pilus (Tcp) in Vibrio cholerae (Taylor et al., 1987). Osbourn, Barber, and Daniels develop a “promoter-probe” plasmid to identify in planta induced genes in Xanthomonas campestri (Osbourn et al., 1987). Falkow puts forth his “Molecular Koch’s Postulates” (Falkow, 1988). Mahan, Slauch, and Mekalanos develop in vivo expression technology (IVET) to identify in vivo induced genes in animal pathogens (Mahan et al., 1993a). This is followed by a series of variations on the IVET theme (see Table 1). Hensel, Shea, Gleeson, Jones, Dalton, and Holden develop signature tagged mutagenesis (STM), which allows negative selection of bacterial genes required during growth in the host (Hensel et al., 1995). The first complete genome sequence of a bacterium, Haemophilus influenzae, is published (Fleischmann et al., 1995). Akerley, Rubin, Camilli, Lampe, Robertson, and Mekalanos introduce genomic analysis and mapping by in vitro transposition (GAMBIT) to identify bacterial genes required under a particular condition (Akerley et al., 1998). Sassetti, Boyd, and Rubin develop transposon site hybridization (TraSH), which uses genomic microarrays and random transposition to identify bacterial gene required under a given condition (Sassetti et al., 2001).

1. Introduction Free-living organisms often adapt to changes in their environment. These can be relatively simple adjustments that allow the organism to use a new carbon source, or a more complex transformation such as a pathogen adapting to

Chapter 1. Genetic Analysis of Bacterial Pathogenesis

3

colonize a particular niche in the host. Indeed, bacterial pathogens must often adapt to multiple host environments in order to survive and propagate—their primary purpose. Pathogens adapt to the host environments by inducing genes that encode virulence factors, which specifically interact with the host to colonize or to circumvent the immune response, as well as metabolic genes appropriate to the particular niche. Many pathogens are capable of surviving and propagating in a variety of host issues, and often regulate gene expression both temporally and spatially. In order to understand the molecular mechanisms of bacterial pathogenesis, one needs to identify the gene products required for adaptation to these host environments. However, the host is usually a “black box.” In other words, we do not understand the host environments, and therefore we cannot mimic these environments in the laboratory in order to ask what genes are regulated in response to, or are required under, these conditions. A primary goal, therefore, in pathogenesis is to identify the genes and gene products that are required for growth and survival in the host. Many investigators have applied the techniques of bacterial genetics that have been so useful in the study of bacteria in the laboratory to bacterial pathogenesis. Select examples will be discussed in this chapter. These techniques were designed for use in various animal models of infection and will be described in this context. However, it should be noted that these techniques simply compare gene expression or gene requirements under two environmental conditions and have applications in areas other than pathogenesis.

2. Fusion-based Techniques for Identification of Virulence Genes 2.1. TnPhoA Studying the regulation of gene expression is simplified by the use of gene fusions. Rather than monitoring the function of individual proteins (often a difficult task), the regulatory signals of the protein are connected to reporter genes whose expression can easily be assayed (Slauch and Silhavy, 1991). Fusions come in two types. In a transcriptional fusion the reporter gene encodes its own translational start site but is dependent on the promoter of the gene of interest for transcription. In a translational fusion, the open reading frame of the gene of interest is fused with the open reading frame of the reporter gene. The result is a hybrid protein where, for example, the amino terminal portion of the hybrid is encoded by the target gene and the carboxy terminal domain is the reporter. In this case, production of the reporter protein is dependent on the transcription, translation, and, perhaps, localization signals encoded by the gene of interest. One of the first applications of fusions to the study of bacterial pathogenesis took advantage of the properties of PhoA translational fusions. The Escherichia coli PhoA protein is an alkaline phosphatase that is normally

4

James M. Slauch

secreted to the periplasmic space. Localization requires recognition of the N-terminal signal sequence by the “sec” secretion system (de Keyzer et al., 2003). The signal sequence is cleaved during export and the mature protein is released into the periplasm. Enzymatic activity of PhoA is dependent on export to the periplasm; the protein will not function in the reducing environment of the cytoplasm (Derman and Beckwith, 1991). Manoil and Beckwith (1985) constructed transposon Tn5 derivatives (see Chapter 2) containing a phoA open reading frame lacking the normal signal sequence. The phoA is positioned in the transposon such that when the element transposes into a gene in the correct orientation and reading frame, translation of the target gene proceeds through phoA producing a hybrid protein (Figure 1). If the target gene is normally localized to an extracytoplasmic location as directed by an N-terminal signal sequence, then the fusion protein will also be exported and the PhoA will be active (Manoil et al., 1990). Thus, one can screen for active PhoA fusions after random transposition into the chromosome and identify extracytoplasmic proteins. Numerous derivatives of TnPhoA have been developed and detailed protocols are available (Manoil, 2000). Taylor et al. (1987) reasoned that some proteins important for interaction between a pathogen and the host would be localized to the surface of the bacterium. They also reasoned that important virulence factors would be coordinately regulated. They knew that the ctxAB operon, which encodes the cholera toxin (CTX), a major virulence factor in Vibrio cholerae, is regulated

Periplasm

Cytoplasm Signal sequence PhoA Cytoplasmic

PhoA Periplasmic or outer membrane

PhoA Integral membrane

FIGURE 1. PhoA fusions identify envelope proteins. Insertion of TnPhoA in frame with a target protein results in production of a hybrid protein. If the hybrid protein is localized to the cytoplasm, PhoA does not fold appropriately and is nonfunctional. If the fusion protein is targeted to the periplasm via an amino-terminal signal sequence or if the fusion joint corresponds to a periplasmic portion of a polytopic inner membrane protein, PhoA is active.

Chapter 1. Genetic Analysis of Bacterial Pathogenesis

5

at the transcriptional level by ToxR in response to certain environmental signals. They randomly transposed the TnPhoA transposon into the chromosome of V. cholerae and subsequently screened active PhoA fusions (fusions to exported proteins) for those that were regulated in the same manner as ctxAB. They thereby identified the “toxin-coregulated pilus” Tcp. We now know that Tcp is a major virulence factor in V. cholerae. The pilus is required for colonization of human intestines and serves as the receptor for the CTX bacteriophage, which encodes the CTX (Davis and Waldor, 2003). Bacterial two-component regulatory systems (see Chapter 13) are conserved proteins that sense environmental signals and respond, often by changing gene expression. Miller et al. (1989) tested the role of a series of two-component systems in the virulence of Salmonella typhimurium and showed that the PhoPQ system was critical in this bacterium. They subsequently screened a series of random PhoA fusions for those that are either induced or repressed when the PhoPQ system was activated. They successfully identified a large number of genes, many of which have subsequently been shown to be important for Salmonella to adapt and grow in macrophages. For example, PhoPQ activates genes whose products alter the integrity of the outer membrane and represses genes encoding a type III secretion system (see Chapter 9) that is required at another stage of infection (Groisman, 2001). Insertion of the TnPhoA into a gene almost certainly disrupts the function of that gene. Thus, one can ask if the gene is important for virulence by testing the insertion mutant in an appropriate infection model. It was shown that many of the PhoPQ-regulated TnPhoA insertions in Salmonella conferred a virulence defect in a mouse model (Belden and Miller, 1994; Miller et al., 1989). However, there is a caveat to this experiment. Although the function of the target gene is almost certainly compromised, a fusion protein is being produced. It is possible that any phenotype conferred is due not to loss of function of the target gene, but rather to a toxic effect of the fusion protein. Indeed, subsequent analysis showed that deletion of some of the genes originally identified as attenuating PhoA fusions in Salmonella did not confer a virulence defect (Gunn et al., 1995; Miller et al., 1992).

2.2. In Vivo Expression Technology In vivo expression technology (IVET) takes advantage of transcriptional gene fusions to directly select for bacterial genes that are transcriptionally active during infection. The selected genes are subsequently screened for those that are transcriptionally inactive during growth in laboratory medium, thus identifying genes that are transcriptionally induced when the pathogen is in the host. A subset of those genes that are induced in host tissues include virulence genes that are specifically required for the infection process. Detailed protocols and reviews are available (Angelichio and Camilli, 2002; Slauch and Camilli, 2000).

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James M. Slauch

Various IVET systems have been used to identify in vivo induced genes in both prokaryotic and eukaryotic pathogens (Table 1). These IVET systems are of three types: (1) selection systems based on metabolic (e.g., purA) or antibiotic (e.g., cat) reporters; (2) recombination-based systems; and (3) green fluorescent protein (GFP) based systems. The IVET selection system was developed in S. typhimurium (Mahan et al., 1993a; Slauch and Camilli, 2000). The original IVET system is based on the fact that mutations in the purA gene, encoding adenylosuccinate synthetase, required for synthesis of adenosine 5′-monophosphate (AMP), dramatically attenuate survival of Salmonella in host tissues (McFarland and Stocker, 1987). In the animal, this provides a means to select for genes that are transcriptionally active in vivo by creating transcriptional fusions to a promoterless purA gene. In order for any given clone to survive and replicate in the host, it must contain a purA fusion to a promoter that is transcriptionally active enough to overcome the parental PurA deficiency. The fusion strains that survive in the host are

TABLE 1. IVET systems and selections.a Organism

Selection/Screen

Reference

Xanthomonas campestri Salmonella typhimurium

cat purA-lacZY (pIVET1)

Vibrio cholerae Listeria monocytogenes S. typhimurium Pseudomonas aeruginosa S. typhimurium

γδ resolvase (pIVET5) lacZ cat-lacZY (pIVET8) purEK GFP

Yersinia entercolitica Escherichia coli Staphylococcus aureus Actinobacillus pleuropneumoniae Streptococcus gordonii P. fluorescens Candida albicans L. monocytogenes P. putida Histoplasma capsulatum Klebsiella pneumoniae S. suis Shigella flexneri P. syringae Porphyromonas gingivalis P. fluorescens Lactobacillus reuteri

cat cat γδ resolvase luxAB-ribBAH amylase-cat panB-lacZY Flp recombinase hly pyrB-lacZ URA5 galU erm cat-lacZ hrcC-uidA tetA-galK dapB-lacZY ermGT-bglM

Osbourn et al. (1987) Mahan et al. (1993a); Slauch et al. (1994) Camilli et al. (1994) Klarsfeld et al. (1994) Mahan et al. (1995) Wang et al. (1996a, b) Valdivia and Falkow (1997); Valdivia and Ramakrishnan (2000) Young and Miller (1997) Khan and Isaacson (1998) Lowe et al. (1998) Fuller et al. (1999) Kilic et al. (1999) Rainey (1999) Staib et al. (1999) Gahan and Hill (2000) Lee and Cooksey (2000) Retallack et al. (2000) Lai et al. (2001) Smith et al. (2001) Bartoleschi et al. (2002) Boch et al. (2002) Wu et al. (2002) Gal et al. (2003) Walter et al. (2003)

a

Listed in chronological order.

Chapter 1. Genetic Analysis of Bacterial Pathogenesis

7

subsequently screened for those that are transcriptionally inactive in laboratory medium. This subset of strains contains fusions to genes that are transcriptionally active only during infection. In other words, these genes are specifically induced in the host. The IVET system was designed with several properties. First, the fusions are constructed in single copy in the chromosome. This avoids any complications that can arise from the use of multicopy plasmids. Second, the fusions are constructed without disruption of any chromosomal genes. If the gene of interest encodes a product required for the infection process, then a fusion that disrupts the gene would not be recovered in the selection. Third, the fusion provides a convenient method to monitor transcriptional activity both in vitro and in vivo. The pIVET1 plasmid is based on the “suicide vector” pGP704. Replication of pGP704 derivatives requires the replication protein, Pi, the product of the pir gene, which must be supplied in trans. Therefore, the plasmid will replicate autonomously in Pi+ strains. In strains lacking Pi, the plasmid must integrate into the chromosome in order to be stably maintained (see below). The plasmid also contains the cis-acting site (mob) that allows broad host-range conjugal transfer by plasmid RP4 (see Chapter 2). The pIVET1 plasmid (Figure 2) contains a synthetic operon composed of a promoterless purA gene followed by a promoterless lac operon, encoding amp

X9

purA

lacZ

lacY

X+

X9

purA

lacZ

lacY

amp

X+

FIGURE 2. Construction of IVET fusions. Random chromosomal fragments are cloned into the IVET vector. Approximately one half of the clones will contain an appropriately positioned promoter that will control expression of the polycistronic purA lacZ lacY operon. Introduction of the resulting plasmid into a Pi− strain where the plasmid cannot replicate and growth in the presence of ampicillin selects for strains in which the plasmid has integrated via homologous recombination. This results in a single-copy fusion controlled by the chromosomal promoter. This strain is also a merodiploid with a wild-type copy of the gene of interest. Note that the background strain is ∆purA.

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James M. Slauch

β-galactosidase (LacZ) and Lac permease (LacY). Upstream of the purA gene is a unique BglII restriction site. Random Sau3AI fragments of S. typhimurium chromosomal DNA, isolated from a ∆purA strain, are cloned into the BglII site, 5′ to the purA gene in pIVET1. This often results in transcriptional fusions in which S. typhimurium promoters drive the expression of a wild-type copy of purA and lacZY. Conjugal introduction of the pool of fusions into a ∆purA strain of S. typhimurium that lacks the pir gene, and selection for resistance to ampicillin, demands the integration of the plasmids into the chromosome by homologous recombination with the cloned Salmonella DNA. In clones that have a promoter in front of purA, this integration event results in single-copy diploid fusions in which one promoter drives the expression of the purA-lac fusion and the other promoter drives the expression of the wild-type gene (Figure 2). The integration event is key to the design of the system. The cloned chromosomal sequences provide the only site of homology between the plasmid and the chromosome. The purA gene is from E. coli, which has diverged sufficiently from S. typhimurium to prevent recombination (Rayssiguier et al., 1989). In addition, S. typhimurium does not possess a lac operon. Note also that only fusion plasmids that contain the promoter from the operon of interest will maintain synthesis of the wild-type gene upon integration (the event drawn in Figure 2). Clones that do not contain a promoter in the correct orientation are unlikely to answer the selection. For example, clones that contain an internal fragment of an operon will generate a fusion under the control of the chromosomal promoter, but will disrupt the expression of the wild-type gene. If the gene encodes a virulence factor, then loss of the gene product could result in selection against this construct in the animal. Likewise, clones that are in the wrong orientation with respect to the fusion will never express purA and will, therefore, be selected against in the animal. In practice, chromosomal DNA is partially digested using the restriction enzyme Sau3AI and the fragments are size-fractionated. These fragments are then cloned into the BglII site in the IVET vector. In the original experiment, fragments of approximately 5 kilobases (kb) were selected (Mahan et al., 1993c). This facilitates integration of the plasmid into the chromosome and increases the likelihood that any clone will contain the promoter region for the gene located at the fusion joint. The number of independent clones should be sufficiently large to ensure a fusion to every gene in the chromosome. How is this determined? The chromosome of S. typhimurium is approximately 4.5 × 106 basepairs (bp) and the average gene is about 1,000 bp. The number of independent clones required to ensure, with 95% confidence, that there is a fusion every 500 bp is approximately 54,000. This is calculated from the formula N = ln(1−P)/ln(1−f), where P is the probability (0.95 in our example) and f is the “fraction of chromosome” (500 bp/4.5 × 106 bp; Clarke and Carbon, 1976). Because there is an equal probability that any given clone will be in the wrong orientation with respect to the fusion joint, the number is multiplied by 2 in this case. Note that this calculation is

Chapter 1. Genetic Analysis of Bacterial Pathogenesis

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based on the interval between fusion joints (500 bp), not on the size of the clones (5 kb). The relative activity of the fusions in the population can be monitored by plating the bacteria on lactose indicator medium, e.g., Lactose MacConkey Agar (Figure 3). At least 50 percent of the clones should be phenotypically Lac−, because half of the time the fusion is in the wrong orientation with respect to the gene. This mixed population of fusion strains can now be used to infect an animal model or incubated under other conditions that potentially mimic a host environment. During Salmonella infection of a mouse, the requirement for PurA activity is absolute, and only those strains that contain active fusions survive, independent of anatomic location. In other words, the selection is as strong in the intestine as it is during systemic infection. Indeed, the selection has been used to identify genes that are transcriptionally active only in particular tissues of the animal (Stanley et al., 2000).

In vivo induced Lac+

In vivo induced Lac−

Lac+

Lactose MacConkey Agar

Preselection population