Neisseria Meningitidis: Advanced Methods and Protocols (Methods in Molecular Biology 799)

METHODS

IN

MOLECULAR BIOLOGY™

Series Editor John M. Walker School of Life Sciences University of Hertfordshire Hatfield, Hertfordshire, AL10 9AB, UK

For further volumes: http://www.springer.com/series/7651

wwwwwwwwwwwww

Neisseria meningitidis Advanced Methods and Protocols

Edited by

Myron Christodoulides Division of Infection, Inflammation, and Immunity, Sir Henry Wellcome Laboratories, University of Southampton Medical School, Southampton, UK

Editor Myron Christodoulides, Ph.D Division of Infection, Inflammation, and Immunity Sir Henry Wellcome Laboratories University of Southampton Medical School Southampton, UK [email protected]

ISSN 1064-3745 e-ISSN 1940-6029 ISBN 978-1-61779-345-5 e-ISBN 978-1-61779-346-2 DOI 10.1007/978-1-61779-346-2 Springer New York Dordrecht Heidelberg London Library of Congress Control Number: 2011937568 © Springer Science+Business Media, LLC 2012 All rights reserved. This work may not be translated or copied in whole or in part without the written permission of the publisher (Humana Press, c/o 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. While the advice and information in this book are believed to be true and accurate at the date of going to press, neither the authors nor the editors nor the publisher can accept any legal responsibility for any errors or omissions that may be made. The publisher makes no warranty, express or implied, with respect to the material contained herein. Printed on acid-free paper Cover illustration: The cover picture is a confocal microscopy image of human meningeal cells (stained red with Evan’s blue) infected with Neisseria meningitidis (bacteria reacted with specific antibody to outer membranes and stained green with FITC-labelling). Image copyright Myron Christodoulides. Humana Press is part of Springer Science+Business Media (www.springer.com)

Preface Neisseria meningitidis (the meningococcus, family Neisseriaceae) is a major causative agent, worldwide, of potentially life-threatening meningitis and septicemia. The organism is a Gram-negative β proteobacterium that resides normally as a commensal on the mucosal epithelium of the human nasopharynx. However, a combination of the expression of key microbial virulence factors, host susceptibility, inadequate innate immune recognition, and changes in niche environmental conditions can favor bacterial invasion of the host. The pathogenesis of meningococcal infection involves initial penetration of the nasopharyngeal mucosal epithelium, entry into the blood, and the development of a bacteremia, which occurs in the absence of host humoral immunity. The meningococcus is a classical meningeal pathogen, i.e., it is capable of passing across the blood-cerebrospinal fluid (CSF) barrier to enter the CSF-filled subarachnoid space (SAS) to colonize the leptomeninges [1–3]. The consequences to the host are a compartmentalized intravascular inflammatory response, septicemia, and/or a compartmentalized intracranial inflammatory response, leptomeningitis [4]. Meningococcal infection can be sporadic, hyper-sporadic, or epidemic in nature, and global estimates for infection are ~1.2 million cases each year with a toll of ~135,000 deaths [5]. Despite successful antibiotic treatment and advances in intensive care management of patients with meningococcal infection, the mortality rate is still high at ~15% in industrialized countries, and the disease is rightly feared by the public, parents, and physicians for the rapidity of onset of both clinical symptoms and the decline of patients’ health. Moreover, survivors are often faced with permanent physical and neurological sequelae, including loss of limbs; auditory and visual impairment; cognitive dysfunction; educational, behavioral, and developmental problems; seizures and persistent headaches; and motor nerve deficits, hydrocephalus, and permanent brain damage [6]. These outcomes impact the quality of life of the affected individuals and their families and on the providers of health, welfare, and social services. Since Weichselbaum first identified the meningococcus from the CSF of a patient with meningitis in 1887 [7], key discoveries about the nature of the organism, the pathogenesis of infection, and the mechanisms of natural immunity have led, finally, to the development and introduction of safe and effective vaccines. The classical studies by Goldschneider and colleagues in the late 1960s identified the correlation between the development of serum bactericidal antibodies and protection from meningococcal infection [8, 9]. Combined with our increased understanding of meningococcal surface antigen structure and function, capsule polysaccharide-protein conjugate vaccines against meningococci that express the serogroup A, C, Y, and W-135 capsules have been developed and introduced into adolescent and adult immunization schedules [10, 11] and trialed in young children [12]. Conjugate vaccines have already significantly reduced meningococcal disease in industrialized countries, e.g., for serogroup C infection [13], and the long-term expectation is that the introduction of new conjugate vaccines against serogroup A will lead to a decline in epidemic disease reported in the “African Meningitis Belt” [14]. Today, a concerted effort is aimed at developing vaccines to serogroup B meningococci, which present a greater technical challenge, due to the poor immunogenicity of its capsule and the molecular mimicry of foetal NCAM, thereby necessitating a search for subcapsular antigens capable of inducing protective

v

vi

Preface

immune responses. The past decade has seen the publication of many Neisseria spp. genomes [15–22], and these have under-pinned advances in molecular methods and techniques with applications to vaccine design. The result is new vaccines for serogroup B that are showing promise in clinical trials [23–25] and a plethora of experimental vaccines that have shown success in preclinical, laboratory studies. The key discoveries about the meningococcus would not have been possible without significant developments in laboratory methods for studying the pathogen at the molecular and cellular levels. Many of these laboratory methods have been described in the landmark books on meningococcal vaccines and meningococcal disease, edited by Andrew Pollard and Martin Maiden for the series Methods in Molecular Medicine [26, 27]. This new book, Neisseria meningitidis: Advanced Methods and Protocols, does not simply revisit and update the methods and protocols described in the books from Pollard and Maiden, a task of itself unnecessary due to the comprehensive nature of these previous volumes, but it offers a collection of advanced methods and protocols that in many ways reflect the development and refinement of several new technologies applied to the meningococcus. Several of the chapters in this book describe methods that rely on the collection of complete sets of biological data, for example, using the genome to generate transcriptomes, proteomes, and metabolomes. However, there are many new -omics that are being developed both theoretically and practically (e.g., the interactome, moleculome, cytome, and regulome, to name but a few), and these are now beginning to be applied to the study of Neisseria and many other human pathogens. Laboratory methods and protocols for these new -omics could be the subjects of future volumes. Neisseria meningitidis: Advanced Methods and Protocols begins with a review of the biology, microbiology, and epidemiology of the meningococcus, which is followed by two chapters that provide a clinical context, namely, in the classification and pathogenesis of meningococcal infections and a technique for detecting the pathogen in CSF samples from patients. In cases of undefined meningeal irritation, the latter method provides a means for identifying between the important bacterial and viral causes of meningitis. A number of chapters then follow that provide methods and protocols for investigating the molecular biology and biochemistry of the meningococcus. These techniques can provide useful tools for vaccine and pathogen–host interaction studies and include methods for generating knock-out and complementation strains of the meningococcus, for identifying and characterizing small RNA molecules and for the expression of purified meningococcal proteins for crystallization. A particular area of Neisseria research that, if not exactly neglected, is not always appreciated relates to the metabolism of the meningococcus. Hence, a chapter is provided that explains how genome-scale metabolic networks can be constructed using a constraint-based modeling approach, using available genome sequence databases and high-throughput bioinformatics. This is followed by a complementary method for studying adaptations in meningococcal/microbial proteomes to changing environmental conditions. The next collection of chapters broadly covers pathogen–host cell interactions and is prefaced with an introductory review of our current knowledge of meningococcal surface ligands and their respective host cell receptors. A protocol for studying meningococcal interactions with an animal model is presented, followed by methods used for culturing and investigating biofilms in vitro. Interactions of bacteria with host cells are subject to environmental stress and physical forces applied to bacterial ligand–host cell receptor binding events, and methods are provided for investigating bacterial adhesion under shear stress and the forces exerted by the meningococcal pilus adhesin. The literature is replete with in vitro cell culture models used for studying bacterial interactions, from human explant models to

Preface

vii

monocultures of primary cells and transformed cells of myeloid and nonmyeloid origins: for this book, protocols are described for isolating human dendritic cells and using them to study host–Neisseria interactions, acknowledging the important role that these cells have in sentinel immune recognition during Neisseria infection and in driving polarization of naïve T-cell helper responses. Finally, the events that follow bacterial interaction with host cell receptors are considered in two chapters that present methods for investigating ligand– receptor interactions, by using hydrogen/deuterium exchange coupled to mass spectrometry and nanoscale imaging techniques to visualize the interactions between pathogen-associated molecular patterns (PAMP) and host pattern recognition receptors (PRR). The next chapters consider the consequences of meningococcal interaction with host cells; in-depth protocols are provided for analyzing the transcriptome of the pathogen and host epithelial and endothelial cell models, followed by a detailed technical review on the experimental design that allows the researcher to generate valuable and reliable data from using the pan-Neisseria microarray. A major consequence of meningococcal infection is host cell damage, clearly seen in patients with sepsis and meningitis, and in vitro methods are provided for investigating host cellular apoptosis/necrosis induced by the pathogenic Neisseriae. The final part of this book focuses on methods and protocols for vaccine antigen discovery and vaccine design. Methods are provided for two different approaches, one using proteomics to analyze the human immune response to Neisseria meningitidis and the other, “reverse vaccinology.” In particular, the latter chapter provides detailed methods for in silico identification and selection of antigens, through production of recombinant proteins for immunization and analyses of the immune response. The final chapter provides a protocol for preparing experimental DNA vaccines to bacterial polypeptides. Many of the techniques described herein can be readily used to study other pathogens and diseases and should have broad appeal to clinical and nonclinical scientists alike. I do accept that some of these methods can seem daunting or require specialized equipment, but I do hope that they stimulate collaboration between readers and authors. This book could not have been possible without the contributions of many, and I would like to express my gratitude toward all authors, all of whom enthusiastically contributed their articles and showed patience with my editing; to the staff at Humana Press for commissioning this volume and especially to the series editor, John Walker, who has provided support and advice when needed. Finally, although this past decade has seen tremendous advances in the fight against meningococcal infection, there is still much to learn about the meningococcus and not only does it continue to surprise us with its complex nature, but also its relationship with its host reveals a great deal about human biology. Southampton, UK

Myron Christodoulides

References 1. Carbonnelle E, Hill DJ, Morand P et al (2009) Meningococcal interactions with the host. Vaccine 27: B78–B89. 2. Christodoulides M, Heckels JE, Weller RO (2002) The role of the leptomeninges in meningococcal meningitis In: Ferreiros C, Criado MT, Vazquez J (ed) Emerging strategies in the fight against meningitis. Horizon Press.

3. Join-Lambert O, Morand PC, Carbonnelle E et al (2010) Mechanisms of meningeal invasion by a bacterial extracellular pathogen, the example of Neisseria meningitidis. Prog Neurobiol 91: 130–139. 4. Brandtzaeg P (1995) Pathogenesis of meningococcal infections. In: Cartwright KAV (ed) Meningococcal Disease. Wiley, New York.

viii

Preface

5. Stephens DS, Greenwood B, Brandtzaeg P (2007) Epidemic meningitis, meningococcaemia, and Neisseria meningitidis. Lancet 369: 2196–2210. 6. Steven N, Wood M (1995) The clinical spectrum of meningococcal disease. In: Cartwright KAV (ed) Meningococcal Disease. Wiley, New York. 7. Weichselbaum A (1887) Ueber die aetiologie der akuten meningitis cerebrospinalis. Fortschr Med 5: 573. 8. Goldschneider I, Gotschlich EC, Artenstein MS (1969) Human immunity to the meningococcus. I. The role of humoral antibodies. J Exp Med 129: 1307–1326. 9. Goldschneider I, Gotschlich EC, Artenstein MS (1969) Human immunity to the meningococcus. II. Development of natural immunity. J Exp Med 129: 1327–1348. 10. Pace D (2010) Novel quadrivalent meningococcal A, C, W-135 and Y glycoconjugate vaccine for the broader protection of adolescents and adults. Future Microbiol 5: 1629–1640. 11. Keyserling HL, Pollard AJ, Detora LM et al (2006) Experience with MCV-4, a meningococcal, diphtheria toxoid conjugate vaccine against serogroups A, C, Y and W-135. Expert Rev Vaccine 5: 445–459. 12. Halperin SA, Gupta A, Jeanfreau R et al (2010) Comparison of the safety and immunogenicity of an investigational and a licensed quadrivalent meningococcal conjugate vaccine in children 2-10 years of age. Vaccine 28: 7865–7872. 13. Borrow R, Miller E (2006) Long-term protection in children with meningococcal C conjugate vaccination: lessons learned. Expert Rev Vaccine 5: 851–857. 14. Okoko BJ, Idoko OT, Adegbola RA (2009) Prospects and challenges with introduction of a mono-valent meningococcal conjugate vaccine in Africa. Vaccine 27: 2023–2029. 15. Tettelin H, Saunders NJ, Heidelberg J et al (2000) Complete genome sequence of Neisseria meningitidis serogroup B strain MC58. Science 287: 1809–1815. 16. Parkhill J, Achtman M, James KD et al (2000) Complete DNA sequence of a serogroup A strain of Neisseria meningitidis Z2491. Nature 404: 502–506.

17. Bentley SD, Vernikos GS, Snyder LAS et al (2007) Meningococcal genetic variation mechanisms viewed through comparative analysis of serogroup C strain FAM18. PloS Genetics 3: 230–240. 18. Peng JP, Zhang XB, Yang E et al (2007) Characterization of serogroup C meningococci isolated from 14 provinces of China during 1966-2005 using comparative genomic hybridization. Sci China Ser C-Life Sciences 50: 1–6. 19. Schoen C, Blom J, Claus H et al (2008) Wholegenome comparison of disease and carriage strains provides insights into virulence evolution in Neisseria meningitidis. Proc Nat Acad Sci USA 105: 3473–3478. 20. Bennett JS, Bentley SD, Vernikos GS et al (2010) Independent evolution of the core and accessory gene sets in the genus Neisseria: insights gained from the genome of Neisseria lactamica isolate 020-06. Bmc Genomics 11:652. 21. Lavezzo E, Toppo S, Barzon L et al (2010) Draft genome sequences of two Neisseria meningitidis serogroup C clinical isolates. J Bact 192: 5270–5271. 22. Marri PR, Paniscus M, Weyand NJ et al (2010) Genome sequencing reveals widespread virulence gene exchange among human Neisseria species. PlosSOne 5:e11835. 23. Rinaudo CD, Telford JL, Rappuoli R et al (2009) Vaccinology in the genome era. J Clin Invest 119: 2515–2525. 24. Findlow J, Borrow R, Snape MD et al (2010) Multicenter, open-label, randomized Phase II controlled trial of an investigational recombinant meningococcal serogroup B vaccine with and without outer membrane vesicles, administered in infancy. Clin Infect Dis 51: 1127–1137. 25. Richmond P, Marshall H, Sheldon E et al (2010) Safety and immunogenicity of serogroup B Neisseria meningitidis (MnB) rLP2086 vaccine in adults and adolescent subjects: overview of 3 clinical trials, in: 17th International Pathogenic Neisseria Conference. 26. Pollard AJ, Maiden MCJ (2001) Meningococcal vaccines: methods and protocols. Humana Press, Totowa. 27. Pollard AJ, Maiden MCJ (2001) Meningococcal disease: methods and protocols. Humana Press, Totowa.

Contents Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

v xi

1 Neisseria meningitidis: Biology, Microbiology, and Epidemiology . . . . . . . . . . . . . .

1

2 3

4

5

6

7 8

9 10 11 12

13 14

Nadine G. Rouphael and David S. Stephens Classification and Pathogenesis of Meningococcal Infections . . . . . . . . . . . . . . . . . . Petter Brandtzaeg and Marcel van Deuren Detection of Neisseria meningitidis in Cerebrospinal Fluid Using a Multiplex PCR and the Luminex Detection Technology. . . . . . . . . . . . . . . . . . . . Jens Kjølseth Møller Generating Knock-Out and Complementation Strains of Neisseria meningitidis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Vincent van Dam and Martine P. Bos Identification and Functional Characterization of sRNAs in Neisseria meningitidis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Yvonne Pannekoek and Arie van der Ende Expression, Purification, and Crystallization of Neisserial Outer Membrane Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Muhammad Saleem, Jeremy Moore, and Jeremy P. Derrick Genome-Scale Metabolic Models: Reconstruction and Analysis . . . . . . . . . . . . . . . . Gino J.E. Baart and Dirk E. Martens TMT Labelling for the Quantitative Analysis of Adaptive Responses in the Meningococcal Proteome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Karsten Kuhn, Christian Baumann, Jan Tommassen, and Thorsten Prinz Meningococcal Ligands and Molecular Targets of the Host. . . . . . . . . . . . . . . . . . . Darryl J. Hill and Mumtaz Virji In Vivo Imaging of Meningococcal Disease Dynamics. . . . . . . . . . . . . . . . . . . . . . . Hong Sjölinder and Ann-Beth Jonsson Methods for Studying Neisseria meningitidis Biofilms . . . . . . . . . . . . . . . . . . . . . . . Michael A. Apicella, Jianqiang Shao, and R. Brock Neil A Laminar-Flow Chamber Assay for Measuring Bacterial Adhesion Under Shear Stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Magali Soyer and Guillaume Duménil Techniques to Measure Pilus Retraction Forces . . . . . . . . . . . . . . . . . . . . . . . . . . . . Nicolas Biais, Dustin Higashi, Magdalene So, and Benoit Ladoux Human Dendritic Cell Culture and Bacterial Infection . . . . . . . . . . . . . . . . . . . . . . Hannah E. Jones, Nigel Klein, and Garth L.J. Dixon

ix

21

37

55

73

91 107

127

143 153 169

185 197 217

x

Contents

15 Hydrogen–Deuterium Exchange Coupled to Mass Spectrometry to Investigate Ligand–Receptor Interactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

16 17

18

19

20

21

22

Jessmi M.L. Ling, Leslie Silva, David C. Schriemer, and Anthony B. Schryvers Visualising PAMP–PRR Interactions Using Nanoscale Imaging. . . . . . . . . . . . . . . . Kathy Triantafilou and Martha Triantafilou Transcriptome Analyses in the Interaction of Neisseria meningitidis with Mammalian Host Cells . . . . . . . . . . . . . . . . . . . . . . . Biju Joseph, Matthias Frosch, Christoph Schoen, and Alexandra Schubert-Unkmeir The Use of the Pan-Neisseria Microarray and Experimental Design for Transcriptomics Studies of Neisseria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Nigel J. Saunders and John K. Davies Analysis of Parameters Associated with Prevention of Cellular Apoptosis by Pathogenic Neisseriae and Purified Porins. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Paola Massari and Lee M. Wetzler Analysis of the Immune Response to Neisseria meningitidis Using a Proteomics Approach . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Jeannette N. Williams, Myron Christodoulides, and John E. Heckels Antigen Identification Starting from the Genome: A “Reverse Vaccinology” Approach Applied to MenB . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Emmanuelle Palumbo, Luigi Fiaschi, Brunella Brunelli, Sara Marchi, Silvana Savino, and Mariagrazia Pizza A DNA Vaccine Strategy for Effective Antibody Induction to Pathogen-Derived Antigens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Jason Rice and Myron Christodoulides

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

237

253

267

295

319

343

361

405 421

Contributors MICHAEL A. APICELLA • The Department of Microbiology, Carver College of Medicine, The University of Iowa, Iowa City, IA, USA GINO J.E. BAART • VIB Department of Plant Systems Biology/Department of Biology, Protistology and Aquatic Ecology, Ghent University, Ghent, Belgium CHRISTIAN BAUMANN • Proteome Sciences R&D, Frankfurt am Main, Germany NICOLAS BIAIS • Department of Biological Sciences, Columbia University, New York, NY, USA MARTINE P. BOS • Department of Molecular Microbiology, Utrecht University, Utrecht, The Netherlands PETTER BRANDTZAEG • Departments of Pediatrics and Medical Biochemistry, University of Oslo, Oslo, Norway BRUNELLA BRUNELLI • Novartis Vaccines and Diagnostics, Siena, Italy MYRON CHRISTODOULIDES • Division of Infection, Inflammation, and Immunity, Sir Henry Wellcome Laboratories, University of Southampton Medical School, Southampton, UK VINCENT VAN DAM • Department of Molecular Microbiology, Utrecht University, Utrecht, The Netherlands JOHN K. DAVIES • Nursing and Health Sciences, Monash University, VIC, Australia JEREMY P. DERRICK • University of Manchester, Manchester, UK MARCEL VAN DEUREN • Department of Internal Medicine and Nijmegen Institute for Infection, Inflammation and Immunity, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands GARTH L.J. DIXON • Department of Microbiology, Camelia Botnar Laboratories, Great Ormond Street Hospital, London, UK GUILLAUME DUMÉNIL • INSERM, U970, Paris Cardiovascular Research Center PARCC, Paris, France; Université Paris Descartes, UMR-S970, Paris, France ARIE VAN DER ENDE • Department of Medical Microbiology, Academic Medical Center, Center for Infection and Immunity, Amsterdam, The Netherlands LUIGI FIASCHI • Novartis Vaccines and Diagnostics, Siena, Italy MATTHIAS FROSCH • Institute for Hygiene and Microbiology, Würzburg, Germany JOHN E. HECKELS • Division of Infection, Inflammation, and Immunity, Sir Henry Wellcome Laboratories, University of Southampton Medical School, Southampton, UK DUSTIN HIGASHI • Department of Immunobiology and the BIO5 Institute, University of Arizona, Tucson, AZ, USA DARRYL J. HILL • School of Cellular and Molecular Medicine, University of Bristol, Bristol, UK HANNAH E. JONES • Infectious Diseases and Microbiology Unit, Institute of Child Health, University College London, London, UK

xi

xii

Contributors

ANN-BETH JONSSON • Department of Genetics, Microbiology, and Toxicology (GMT), Stockholm University, Stockholm, Sweden BIJU JOSEPH • Institute for Hygiene and Microbiology, Würzburg, Germany NIGEL KLEIN • Infectious Diseases and Microbiology Unit, Institute of Child Health, University College London, London, UK KARSTEN KUHN • Proteome Sciences R&D, Frankfurt am Main, Germany BENOIT LADOUX • Matières et Systèmes Complexes, CNRS UNR7057/Université Paris 7, Paris, France JESSMI M.L. LING • Department of Microbiology and Infectious Diseases, University of Calgary, Calgary, AB, Canada SARA MARCHI • Novartis Vaccines and Diagnostics, Siena, Italy DIRK E. MARTENS • Bioprocess Engineering Group, Wageningen University, Wageningen, The Netherlands PAOLA MASSARI • Department of Medicine, Evans BioMedical Research Center, Section of Infectious Diseases, Boston University School of Medicine, Boston, MA, USA JENS KJØLSETH MØLLER • Department of Clinical Microbiology, Vejle Hospital, Vejle, Denmark JEREMY MOORE • Centre for Structural Biology, Imperial College London, London, UK R. BROCK NEIL • The Department of Microbiology, Carver College of Medicine, The University of Iowa, Iowa City, IA, USA EMMANUELLE PALUMBO • Novartis Vaccines and Diagnostics, Siena, Italy YVONNE PANNEKOEK • Department of Medical Microbiology, Academic Medical Center, Center for Infection and Immunity, Amsterdam, The Netherlands MARIAGRAZIA PIZZA • Novartis Vaccines and Diagnostics, Siena, Italy THORSTEN PRINZ • Proteome Sciences R&D, Frankfurt am Main, Germany JASON RICE • Division of Cancer Sciences, Genetic Vaccines Group, University of Southampton Medical School, Southampton, UK NADINE G. ROUPHAEL • Department of Medicine, Division of Infectious Diseases, Emory University School of Medicine, Atlanta, GA, USA MUHAMMAD SALEEM • University of Manchester, Manchester, UK NIGEL J. SAUNDERS • Sir William Dunn School of Pathology, University of Oxford, Oxford, UK SILVANA SAVINO • Novartis Vaccines and Diagnostics, Siena, Italy CHRISTOPH SCHOEN • Institute for Hygiene and Microbiology, Würzburg, Germany DAVID C. SCHRIEMER • Department of Biochemistry and Molecular Biology, University of Calgary, Calgary, AB, Canada ANTHONY B. SCHRYVERS • Department of Microbiology and Infectious Diseases, University of Calgary, Calgary, AB, Canada ALEXANDRA SCHUBERT-UNKMEIR • Institute for Hygiene and Microbiology, University of Würzburg, Würzburg, Germany JIANQIANG SHAO • The Department of Microbiology, Carver College of Medicine, The University of Iowa, Iowa City, IA, USA LESLIE SILVA • Department of Biochemistry and Molecular Biology, University of Calgary, Calgary, AB, Canada HONG SJÖLINDER • Department of Genetics, Microbiology, and Toxicology (GMT), Stockholm University, Stockholm, Sweden

Contributors

xiii

MAGDALENE SO • Department of Immunobiology and the BIO5 Institute, University of Arizona, Tucson, AZ, USA MAGALI SOYER • INSERM, U970, Paris Cardiovascular Research Center PARCC, Paris, France; Université Paris Descartes, UMR-S970, Paris, France DAVID S. STEPHENS • Department of Medicine, Division of Infectious Diseases, Emory University School of Medicine, Atlanta, GA, USA; Laboratories of Microbial Pathogenesis, Atlanta, VA Medical Center, Decatur, GA, USA JAN TOMMASSEN • Department of Molecular Microbiology, Utrecht University, Utrecht, The Netherlands KATHY TRIANTAFILOU • Department of Child Health, School of Medicine, University Hospital of Wales, Cardiff University, Cardiff, UK MARTHA TRIANTAFILOU • Department of Child Health, School of Medicine, University Hospital of Wales, Cardiff University, Cardiff, UK MUMTAZ VIRJI • School of Cellular and Molecular Medicine, University of Bristol, Bristol, UK LEE M. WETZLER • Department of Medicine, Evans BioMedical Research Center, Section of Infectious Diseases, Boston University School of Medicine, Boston, MA, USA JEANNETTE N. WILLIAMS • Division of Infection, Inflammation, and Immunity, Sir Henry Wellcome Laboratories, University of Southampton Medical School, Southampton, UK

wwwwwwwww

Chapter 1 Neisseria meningitidis: Biology, Microbiology, and Epidemiology Nadine G. Rouphael and David S. Stephens Abstract Neisseria meningitidis (the meningococcus) causes significant morbidity and mortality in children and young adults worldwide through epidemic or sporadic meningitis and/or septicemia. In this review, we describe the biology, microbiology, and epidemiology of this exclusive human pathogen. N.meningitidis is a fastidious, encapsulated, aerobic gram-negative diplococcus. Colonies are positive by the oxidase test and most strains utilize maltose. The phenotypic classification of meningococci, based on structural differences in capsular polysaccharide, lipooligosaccharide (LOS) and outer membrane proteins, is now complemented by genome sequence typing (ST). The epidemiological profile of N. meningitidis is variable in different populations and over time and virulence of the meningococcus is based on a transformable/plastic genome and expression of certain capsular polysaccharides (serogroups A, B, C, W-135, Y and X) and non-capsular antigens. N. meningitidis colonizes mucosal surfaces using a multifactorial process involving pili, twitching motility, LOS, opacity associated, and other surface proteins. Certain clonal groups have an increased capacity to gain access to the blood, evade innate immune responses, multiply, and cause systemic disease. Although new vaccines hold great promise, meningococcal infection continues to be reported in both developed and developing countries, where universal vaccine coverage is absent and antibiotic resistance increasingly more common. Key words: Neisseria meningitidis, Pathogenesis, Bacterial infections, Microbiology, Epidemiology

1. Introduction In 1887, Weichselbaum (1) was the first to identify the meningococcus from the cerebrospinal fluid (CSF) of a patient with meningitis. Epidemics of meningococcal meningitis were first described during the early nineteenth century, in 1805 in Geneva, Switzerland by Vieusseux (2), in 1806 in New Bedford, Massachusetts by Danielson and Mann (3) and in the early 1900s in the African meningitis belt (4). The meningococcus was recognized as a habitant

Myron Christodoulides (ed.), Neisseria meningitidis: Advanced Methods and Protocols, Methods in Molecular Biology, vol. 799, DOI 10.1007/978-1-61779-346-2_1, © Springer Science+Business Media, LLC 2012

1

2

N.G. Rouphael and D.S. Stephens

of the nasopharynx of healthy individuals (5), especially seen in the setting of military recruits camps (6) at the beginning of the twentieth century. Treatment for meningococcal disease included serum therapy introduced in 1913 by Flexner (7) and sulfonamides introduced in 1937 (8). The emergence of resistance to sulfonamides (9) in the 1960s prompted the development of the first vaccines against meningococci (10). Despite an understanding of the pathogenesis, the availability of therapeutic and prophylactic antibiotics and immunizations against important serogroups, the meningococcus remains a leading cause worldwide of bacterial meningitis (11). Invasive meningococcal disease results from the interplay of: (1) microbial factors influencing the virulence of the organism, (2) environmental conditions facilitating exposure and acquisition, and (3) host susceptibility factors favoring bacterial acquisition, colonization, invasion, and survival. In the pre-serum therapy and pre-antibiotic eras, 70–85% of meningococcal disease cases were fatal; today, the overall mortality rate in invasive meningococcal disease still remains high, at between 10 and 15% (12). Meningococcal disease is also associated with marked morbidity including limb loss, hearing loss, cognitive dysfunction, visual impairment, educational difficulties, developmental delays, motor nerve deficits, seizure disorders, and behavioral problems (13). In this chapter, we review the biology, microbiology, and epidemiology of the meningococcus.

2. Biology of the Meningococcus The virulence (14) of N. meningitidis is influenced by multiple factors: capsule polysaccharide expression, expression of surface adhesive proteins (outer membrane proteins including pili, porins PorA and B, adhesion molecules Opa and Opc), iron sequestration mechanisms, and endotoxin (lipooligosaccharide, LOS). N. meningitidis also has evolved genetic mechanisms resulting in a horizontal genetic exchange, high frequency phase, antigenic variation, and molecular mimicry, allowing the organism to successfully adapt at mucosal surfaces and invade the host (14). 2.1. Genetics

Genome sequences for a number of N. meningitidis strains including MC58 (serogroup B, ST-32) (15), Z2491 (serogroup A, ST-4) (16), FAM18 (serogroup C, ST-11), and NMB-CDC (serogroup B, ST-8) have been reported. Based on the sequencing of several genomes, the chromosome is between 2.0 and 2.2 megabases in size and contains about 2,000 genes (17). Except for the IHT-A1 capsule locus, no specific core pathogenome has been identified, suggesting that virulence may be clonal group-dependent. The core meningococcal genome that encodes for essential metabolic functions

1 Neisseria meningitidis: Biology, Microbiology, and Epidemiology

3

represents about 70% of the genome. Large genetic islands are present in different strains and are predicted to encode hypothetical surface proteins and virulence factors. The IHT-A1 locus contains the genes for capsule biosynthesis and transport, the IHT-A2 locus is predicted to encode an ABC transporter and a secreted protein, and the IHT-C locus is predicted to encode 30 open reading frames including toxin homologues, a bacteriophage, and potential virulence proteins (18). The meningococcus shares about 90% homology at the nucleotide level with either N. gonorrhoeae or N. lactamica. Mobile genetic elements including IS elements and prophage sequences make up to ~10% of the genome. Transfer of DNA occurs between meningococci, gonococci, and commensal Neisseria spp. as well as other bacteria (such as Haemophilus) (19). Several repetitive sequence and polymorphic regions are present, usually in large heterogeneous arrays, suggesting active areas of genetic recombination. Another characteristic of the meningococcal genome is the presence of multiple genetic switches (e.g., slipped-strand misparing, IS element movement), contributing to the expression of pathogen-associated genes (20). In summary, a central characteristic in the evolution of the meningococcus is the plasticity of the genome and the capacity created by this plasticity for diversity of phenotype. 2.2. Capsule

N. meningitidis can be either encapsulated or not. However, N. meningitidis strains causing invasive disease and isolated from sterile sites such as the blood or the CSF are almost always encapsulated. The capsule is essential for the survival of the organism in the blood as it provides resistance to antibody/complement-mediated killing and inhibits phagocytosis (21). Antibodies directed at capsule play a major part in protection against meningococcal disease and capsule forms the basis for licensed polysaccharide (22, 23) and new conjugate-polysaccharide meningococcal vaccines (24) (except for serogroup B) and for the classification of meningococci into serogroups. The main meningococcal capsular polysaccharides associated with invasive disease are composed of sialic acid derivatives, except for the serogroup A capsule, which consists of repeating units of N-acetyl-mannosamine-1-phosphate. In N. meningitidis, N-acetylneuraminic acid (Neu5Ac), unlike in mammalian cells, is synthesized from N-acetylmannosamine (ManNAc) and phospoenolpyruvate without phosphorylated intermediates (25). Neu5Ac is the most common form of sialic acid in humans and plays an important role in intercellular and/or intermolecular recognition (26). The incorporation of Neu5Ac into meningococcal capsules allows the meningococcus to become less visible to the host immune system (27, 28) because of molecular mimicry. The most striking example is observed in the serogroup B capsule (29), an A(2–8)-linked sialic acid homopolymer identical in structure to the

4

N.G. Rouphael and D.S. Stephens

human fetal neural cell-adhesion molecule (NCAM). Such identity is responsible for the particularly poor immune response generated against serogroup B capsule by humans (30). Capsular genes are located in a single locus cps within the IHTA1 24 kb virulence island that is divided in three regions A, B, and C (31). Region A contains genes responsible for the synthesis and the polymerization of the polysaccharide. Regions B and C contain genes responsible for translocation of the polysaccharide from the cytoplasm to the surface. “Capsule switching” occurs due to genetic identity of parts of the capsule loci and is the result of horizontal exchange by transformation and recombination in the locus of serogroup specific capsule biosynthesis genes (32). Capsule switching is another mechanism of escape from vaccine-induced or natural protective immunity and a virulence mechanism shown by other encapsulated bacterial pathogens (e.g., Streptococcus pneumoniae). For example, the emergence of W-135 as a cause of outbreaks in 2000–2002 was due to the spread of W-135 ET-37 (ST-11) strains closely related to ET-37 (ST-11) serogroup C strains (33, 34). The phenomenon of capsule switching (32, 35) has raised concerns about the immune pressure that capsule-based vaccination programs may apply. However, so far, capsule switching has not caused significant problems after meningococcal conjugate vaccine introduction (36, 37). 2.3. Cell Envelope

In gram-negative bacteria, such as N. meningitidis, the subcapsular cell envelope consists of an outer membrane (OM), a peptidoglycan layer, and a cytoplasmic or inner membrane (Fig. 1). The OM has an outside layer primarily composed of lipopolysaccharide (LPS), actually a lipooligosaccharide, and proteins and an inside layer composed of phospholipids (38) that contains proteins primarily responsible for regulating the flow of nutrients and metabolic products. The major phospholipid component of Neisseria membranes consists largely of phosphatidylethanolamine (PE), with varying amounts of phosphatidylglycerol (PG), cardiolipin (CL), and phosphatidate (PA) (39). The structure of peptidoglycan of different N. meningitidis strains consists of a maximum of two layers (40) with different variations in the degree of cross-linking and O-acetylation. Typically, the percentage of cross-linking of the meningococcal peptidoglycan is around 40%, similar to other gram-negative bacteria (41). The O-acetylation of peptidoglycan results in resistance to lysozyme and to other muramidases (42). Also, peptidoglycan structures are recognized by components of the innate immune system (43).

2.4. Lipopoly saccharide (LPS)

Meningococcal LPS or LOS (endotoxin) (44, 45) plays a role in the adherence of the meningococcus (46) and in activation of the innate immune system (47). Meningococcal LOS lacks a repeating O-side chain of LPS found in enteric gram-negative bacilli (46). Meningococcal LPS consists of three parts (48): lipid A containing

1 Neisseria meningitidis: Biology, Microbiology, and Epidemiology

5

Fig. 1. Cross-sectional view of the meningococcal cell membrane. (Copyright© 2001 Massachusetts Medical Society. All rights reserved).

hydroxy fatty-acid chains and phosphoethanolamine, a core oligosaccharide containing 3-deoxy-D-manno-oct-2-ulosonic acid (KDO) and heptose residues, and highly variable short oligosaccharides. The heptose residues provide linkage to the short oligosaccharide residues of the A-, B-, and G-chains. Meningococcal lipid A, a disaccharide of pyranasol N-acetyl glucosamine residues, is responsible for much of the biological activity and toxicity of meningococcal endotoxin. Sialic acid can be a terminal component of the A-chain lacto-N-neotetraose and is available from endogenous (sialic acid production) or exogenous (host sialic acid at the cell surfaces) sources. The A-chain structures of meningococcal LOS can mimic the human I and i antigens, an example of host molecular mimicry and immune escape mechanisms (49). Phase and antigenic variations (50), leading to different chain oligosaccharide and inner core composition, dramatically alter the antigenic properties of LOS and form the basis of the classification into different immunotypes

6

N.G. Rouphael and D.S. Stephens

(L1-12) (35, 51). Immunotyping screenings of strains from invasive disease and from carriage have indicated that carriage isolates commonly expressed shorter structures termed L1 and L8, while invasive isolates were characterized by the expression of LOS with long A-chains. Also, ~97% of isolates from a serogroup B epidemic reported in England expressed the L3, 7, and 9 (36) immunotype, but the immunotypes of carriers were more heterogeneous. Meningococcal LOS binds to a series of host transfer molecules and receptors on monocytic and dendritic cells of the innate immune system, including LPS-binding protein (LBP), CD14, and myeloid differentiation protein 2 (MD2), part of the Toll-like receptor 4 (TLR4) (52, 53) complex. This triggers the secretion of various cytokines (54) (including IL-6 and TNF-A ) that at high levels can result in endothelial damage and capillary leakage. There is a direct correlation between LPS levels and severity of meningococcal disease (55, 56). LPS also induces the release of chemokines, reactive oxygen species (ROS), and nitric oxide (NO). In addition, LOS also plays an important role in resistance to other host defenses. Meningococci are resistant to cationic antimicrobial peptides (CAMPs) due to the lipid A phosphoethanolamine structures present on lipid A head groups. CAMPs are present in macrophages and neutrophils, and occasionally produced by epithelial cells at mucosal surfaces. These peptides play an important role in host defense against microbial infection and are key components of the innate immune responses through their nonoxidative killing action and their signaling functions. 2.5. Adhesins

Acquisition of meningococci through exposure to respiratory secretions and attachment on human upper respiratory mucosal surfaces by N. meningitidis are the first steps in establishing a human carrier state and invasive meningococcal disease. Meningococcal carriage occurs in 8–25% (57–59) of the human population with adolescents being the major reservoir. Duration of carriage can vary from days to several months. Meningococcal transmission among humans occurs largely through large respiratory droplets; the acquisition may be asymptomatic or may result in local inflammation. Invasive disease usually occurs 1–14 days after acquisition. Multiple structures, termed major and minor adhesions, facilitate meningococcal adherence.

2.5.1. Major Adhesins

Pili: The adhesive properties of capsulate N. meningitidis are mediated by pili (60), which extend several thousand nm beyond the capsule and initiate binding to epithelial cells (61). Twitching motility generated by pilus retraction is important for passage through the epithelial mucus layer, movement over epithelial surfaces, and microcolony formation (62). Piliated meningococci attach to human nasopharyngeal cells (63) in greater numbers

1 Neisseria meningitidis: Biology, Microbiology, and Epidemiology

7

when compared to meningococci devoid of pili. In addition, pili are involved in facilitating the uptake of DNA by meningococci (64) and also enable adherence to endothelial cells and erythrocytes. Meningococcal pili (60) are composed of two major pilin families and undergo both phase and antigenic variation. Neisserial pili can undergo posttranscriptional modifications such as pilin glycosylation (65). Glycosylation (66) may promote secretion of the soluble pilin units that compete for both anti-pili antibodies and host cell receptors allowing protection of the organism. Opacity proteins: N. meningitidis strains commonly express two types of OM opacity proteins, Opa and Opc. While Opc is only expressed by N. meningitidis and encoded by a single gene, Opa proteins are expressed by both meningococci and gonococci, and encoded by multiple genes. Opa expression is subject to antigenic and phase variation and certain Opa types may predominate in clinical isolates due to their adhesion/virulence properties (67). Opa interacts with multiple members of the CEACAM (carcinoembryonic antigen-related cell-adhesion molecule) family (68); during inflammation high levels of CEACAM are expressed, facilitating Opa interactions and therefore cellular attachment and invasion. Both Opa and Opc (69) also interact with cell-surface associated HSPGs (heparan sulfate proteoglycans) (70). 2.5.2. Minor Adhesins

These molecules are often expressed at low levels in vitro, but may be upregulated in vivo; their potential roles in pathogenesis are not fully defined. Examples of minor adhesins include NadA (neisserial adhesinA), NhhA (Neisseria hia homologue A), App (adhesion and penetration protein), and MspA (meningococcal serine protease A) (71).

2.6. Other Molecules

Iron-binding proteins enable meningococci to acquire iron, a crucial growth factor during colonization and disease (72, 73). Meningococcal iron-acquiring proteins include HmbR (hemoglobin), TbpA and TbpB (transferrin), HbpA and HbpB (lactoferrin), HpnA and HpnB (hemoglobin-haptoglobin complex), and possible siderophore homologues. N. meningitidis expresses two distinct porins, PorA and PorB, through which small hydrophilic nutrients diffuse into the bacterium via cation or anion selection. OM porins are also involved in host cell interactions and as targets for bactericidal antibodies (74). PorB is the major OM porin that inserts in membranes, induces Ca2+ influx and activates TLR2 and cell apoptosis (75). PorA is a major component of OM vesicle-based vaccines and a target for bactericidal antibodies (76). Of note, with no universal vaccine for serogroup such as PorA and in other proteins B, there is a particular interest in non-capsular OM antigens identified as vaccine targets through several experimental approaches including “reverse vaccinology” (77).

8

N.G. Rouphael and D.S. Stephens

3. Microbiology of the Meningococcus

3.1. Classification

N. meningitidis is a gram-negative B proteobacterium and member of the bacterial family of Neisseriaceae. N. meningitidis is a fastidious bacterium, dying within hours on inanimate surfaces, and is either an encapsulated or unencapsulated, aerobic diplococcus with a “kidney” or “coffee-bean” shape (Fig. 2). Optimal growth for the organism occurs at 35–37 °C with 5–10% (v/v) carbon dioxide. The organism grows on different media such as blood agar, trypticase soy agar, supplemented chocolate agar, and Mueller-Hinton agar. N. meningitidis colonies on blood agar are grayish, nonhemolytic, round, convex, smooth, moist, and glistening with a clearly defined edge. N. meningitidis, however, tends to undergo rapid autolysis in stationary phase. Colonies are positive by the oxidase test and the result is confirmed with carbohydrate reactions (meningococci oxidize glucose and usually maltose, but not sucrose and lactose). Meningococci are classified according to serological typing (78, 79) and serogrouping is the traditional approach. Further classification into serosubtype, serotype, and immunotype is based on PorA, PorB, and LOS structure, respectively (13). At least 13 distinct

Fig. 2. Intracellular gram-negative diplococci and leukocytes in the CSF from a patient with meningococcal meningitis. The arrow denotes diplococci in proximity and within leukocytes. (Copyright© 2001 Massachusetts Medical Society. All rights reserved).

1 Neisseria meningitidis: Biology, Microbiology, and Epidemiology

9

meningococcal groups have been defined on the basis of their immunological reactivity and structure of the capsule’s polysaccharide (80). These serogroups are the following: A, B, C, E-29, H, I, K, L, W-135, X, Y, Z, and Z’ (29E). Only six serogroups (A, B, C, W-135, X, Y) cause life-threatening disease. Serogroup identification is done by slide agglutination or polymerase chain reaction (PCR) assays, while other meningococcal typing is performed using monoclonal antibodies (mAbs), PCR, and DNA sequencing (81). 3.2. Molecular Typing

Molecular typing is now the preferred approach for identifying clonal groups, closely related strains, strains with the potential to cause outbreaks, and in predicting vaccine coverage and understanding the genome of N. meningitidis. Molecular typing has used multiple techniques (82) including pulsed-field gel electrophoresis (PFGE) (83), multilocus enzyme electrophoresis (MLEE) (84), multilocus sequence typing (MLST) (85), and PCR (86). Currently, MLST is the gold standard for molecular typing (85) and classifies meningococcal strains into different STs (sequence types) based upon polymorphisms in seven housekeeping genes. Many meningococcal STs have been identified, which are independent of the serogroup. Of these, some are disproportionately associated with disease relative to carriage levels and so have been termed hyperinvasive lineages (87). Multilocus and antigen sequence typing data are assembled in large databases accessible via the internet (81).

3.3. Antimicrobial Susceptibility

Antimicrobial susceptibility testing of N. meningitidis should not be performed by disk diffusion, but by either minimal inhibitory concentration (MIC) determination by broth microdilution, or by use of the Etest® strip. To date, antibiotic resistance except for sulfonamides is relatively uncommon in the meningococcus. However, emergence of ciprofloxacin resistant strains (88) was recently observed and a decrease in penicillin susceptibility has been seen with some strains. Laboratory personnel at risk for exposure to aerosolized N. meningitidis should ensure their protective vaccination status is current, and they should work in a biological safety cabinet. Researchers who manipulate invasive N. meningitidis isolates in a manner that could induce aerosolization or droplet formation (i.e., plating, subculturing and serogrouping) on an open bench top and in the absence of effective protection from droplets or aerosols should consider antimicrobial chemoprophylaxis.

3.4. Metabolism

Very little direct work on the metabolism of the meningococcus has been carried out in the past 25 years. In 2007, Bart et al (89) screened the genome of MenB for open reading frames (ORFs) that code for enzymes present in the primary metabolism, yielding a genome-scale metabolic network (90, 91). Their genome-scale flux model was verified using flux balance analysis. According to the model, glucose can be completely catabolized mainly through

10

N.G. Rouphael and D.S. Stephens

the Entner-Douderoff pathway (ED), but also through the pentose phosphate pathway (PP), but not the Embden-Meyerhof-Parnas glycolytic pathway (EMP). The ED cleavage synthesizes the major part of pyruvate (67–87%) (92) and the PP pathway accounts for the remainder. N. meningitidis requires glucose, pyruvate, or lactate as sole carbon source (93) with a certain level of environmental CO2 tension to initiate growth (94). During cultivation on any of the carbon sources, secretion of acetate into the medium occurs (95). Studies on lactate utilization showed that lactate can be utilized by different meningococcal lactate dehydrogenases (LDH) (96). All biochemical pathways for amino acid synthesis in N. meningitidis are available (97). Reduced sulfur in the form of cysteine, cystine, or thiosulfate is required by the meningococcus for growth (98).

4. Epidemiology of the Meningococcus

Meningococcal infection is a global but not uniform problem occurring as sporadic, hyper-sporadic, and epidemic disease (Fig. 3). There are an estimated 1.2 million cases of meningococcal infection per year, with a death toll of ~135,000 worldwide. Disease patterns vary widely over time and between geographical areas, age groups, and bacterial serogroups. Most disease is caused by a few genetically defined clonal complexes of N. meningitidis that can emerge and spread worldwide (85).

Global Serogroup Distribution

" #

! " #

" # 9 ! " # ! 7  # 8 " #

" # "

Fig. 3. Worldwide serogroup distribution of invasive meningococcal disease. (FEMS Microbiol Rev; used with permission).

1 Neisseria meningitidis: Biology, Microbiology, and Epidemiology

4.1. Geographical Areas

11

1. United States: large serogroup A outbreaks occurred in the US during the first part of the twentieth century, but since the 1950s serogroup A meningococcal disease outbreaks disappeared in the USA as well as other industrialized countries (13) for unknown reasons. In the USA, the attack rate is now less than one case per 100,000 per year. During the 10-year period of 1998–2007 and according to the Active Bacterial Core surveillance (ABCs), the annual incidence decreased by 64.1%, from 0.92 cases per 100,000 in 1998 to 0.33 cases per 100,000 in 2007 with an average of 0.53 cases per 100,000 per year (99). A decrease in racial disparities was observed as well. The decrease in rates could not be readily explained by a change in environmental factors, such as smoking (100) and crowding, which are known to be risk factors for meningococcal disease. The decrease in rates was observed prior to implementation of the quadrivalent (serogroups A, C, Y, and W-135) meningococcal conjugate vaccine in 2005 in the USA, which for several reasons had low vaccine uptake in the early years, 11.4% uptake in 2006 and 32.4% uptake in 2007 (99). Infants aged 8)-linked polysialic acid capsule and lipooligosaccharide structure both contribute to the ability of serogroup B Neisseria meningitidis to resist the bactericidal activity of normal human serum. Infect Immun 66:5939–47. 29. Hobb RI, Tzeng YL, Choudhury BP et al (2010) Requirement of NMB0065 for connecting assembly and export of sialic acid capsular polysaccharides in Neisseria meningitidis. Microbes Infect 12:476–87. 30. Zimmer SM, Stephens DS (2006) Serogroup B meningococcal vaccines. Curr Opin Investig Drugs 7:733–9. 31. Frosch M, Weisgerber C, Meyer TF (1989) Molecular characterization and expression in Escherichia coli of the gene complex encoding the polysaccharide capsule of Neisseria meningitidis group B. Proc Natl Acad Sci USA 86:1669–73. 32. Swartley JS, Marfin AA, Edupuganti S, et al (1997) Capsule switching of Neisseria meningitidis. Proc Natl Acad Sci USA 94:271–6. 33. Aguilera JF, Perrocheau A, Meffre C et al (2002) Outbreak of serogroup W135 meningococcal disease after the Hajj pilgrimage, Europe, 2000. Emerg Infect Dis 8:761–7. 34. Raghunathan PL, Jones JD, Tiendrebeogo SR et al (2006) Predictors of immunity after a major serogroup W-135 meningococcal disease epidemic, Burkina Faso, 2002. J Infect Dis 193:607–16. 35. Harrison LH, Shutt KA, Schmink SE et al (2010) Population structure and capsular switching of invasive Neisseria meningitidis isolates in the pre-meningococcal conjugate vaccine era-United States, 2000-2005. J Infect Dis 201:1208–24. 36. Alonso JM, Gilmet G, Rouzic EM et al (2007) Workshop on vaccine pressure and Neisseria meningitidis, Annecy, France, 9–11 March 2005. Vaccine 25:4125–9.

1 Neisseria meningitidis: Biology, Microbiology, and Epidemiology 37. Balmer P, Borrow R, Miller E (2002) Impact of meningococcal C conjugate vaccine in the UK. J Med Microbiol 51:717–22. 38. Nikaido H (1999) Microdermatology: cell surface in the interaction of microbes with the external world. J Bacteriol 181:4–8. 39. Rahman MM, Kolli VS, Kahler CM et al (2000) The membrane phospholipids of Neisseria meningitidis and Neisseria gonorrhoeae as characterized by fast atom bombardment mass spectrometry. Microbiology 146 (Pt 8):1901–11. 40. Antignac A, Rousselle JC, Namane A et al (2003) Detailed structural analysis of the peptidoglycan of the human pathogen Neisseria meningitidis. J Biol Chem 278:31521–8. 41. Quintela JC, Caparros M, de Pedro MA (1995) Variability of peptidoglycan structural parameters in gram-negative bacteria. FEMS Microbiol Lett 125:95–100. 42. Clarke AJ, Dupont C (1992) O-acetylated peptidoglycan: its occurrence, pathobiological significance, and biosynthesis. Can J Microbiol 38:85–91. 43. Girardin SE, Boneca IG, Viala J et al (2003) Nod2 is a general sensor of peptidoglycan through muramyl dipeptide (MDP) detection. J Biol Chem 278:8869–72. 44. Jennings HJ, Johnson KG, Kenne L (1983) The structure of an R-type oligosaccharide core obtained from some lipopolysaccharides of Neisseria meningitidis. Carbohydr Res 121:233–41. 45. Gamian A, Beurret M, Michon F et al (1992) Structure of the L2 lipopolysaccharide core oligosaccharides of Neisseria meningitidis. J Biol Chem 267:922–5. 46. Kahler CM, Stephens DS (1998) Genetic basis for biosynthesis, structure, and function of meningococcal lipooligosaccharide (endotoxin). Crit Rev Microbiol 24:281–334. 47. Plant L, Sundqvist J, Zughaier S et al (2006) Lipooligosaccharide structure contributes to multiple steps in the virulence of Neisseria meningitidis. Infect Immun 74:1360–7. 48. Zughaier SM, Lindner B, Howe J et al (2007) Physicochemical characterization and biological activity of lipooligosaccharides and lipid A from Neisseria meningitidis. J Endotoxin Res 13:343–57. 49. Mandrell RE, Griffiss JM, Macher BA (1988) Lipooligosaccharides (LOS) of Neisseria gonorrhoeae and Neisseria meningitidis have components that are immunochemically similar to precursors of human blood group antigens. Carbohydrate sequence specificity of the mouse monoclonal antibodies that recognize

50.

51.

52.

53.

54.

55.

56.

57.

58.

59.

60.

61.

62.

17

crossreacting antigens on LOS and human erythrocytes. J Exp Med 168:107–26. Jennings MP, Srikhanta YN, Moxon ER et al (1999) The genetic basis of the phase variation repertoire of lipopolysaccharide immunotypes in Neisseria meningitidis. Microbiology 145:3013–21. Mandrell RE, Zollinger WD (1977) Lipopolysaccharide serotyping of Neisseria meningitidis by hemagglutination inhibition. Infect Immun 16:471–5. Zughaier SM, Tzeng YL, Zimmer SM et al (2004) Neisseria meningitidis lipooligosaccharide structure-dependent activation of the macrophage CD14/Toll-like receptor 4 pathway. Infect Immun 72:371–80. Zughaier S, Steeghs L, van der Ley P et al (2007) TLR4-dependent adjuvant activity of Neisseria meningitidis lipid A. Vaccine 25:4401–9. Braun JM, Blackwell CC, Poxton IR et al (2002) Proinflammatory responses to lipooligosaccharide of Neisseria meningitidis immunotype strains in relation to virulence and disease. J Infect Dis 185:1431–8. Brandtzaeg P, Kierulf P, Gaustad P et al (1989) Plasma endotoxin as a predictor of multiple organ failure and death in systemic meningococcal disease. J Infect Dis 159:195–204. Brandtzaeg P, Bryn K, Kierulf P et al (1992) Meningococcal endotoxin in lethal septic shock plasma studied by gas chromatography, mass-spectrometry, ultracentrifugation, and electron microscopy. J Clin Invest 89: 816–23. Greenfield S, Sheehe PR, Feldman HA (1971) Meningococcal carriage in a population of “normal” families. J Infect Dis 123:67–73. Stephens DS (1999) Uncloaking the meningococcus: dynamics of carriage and disease. Lancet 353:941–2. Caugant DA, Hoiby EA, Magnus P et al (1994) Asymptomatic carriage of Neisseria meningitidis in a randomly sampled population. J Clin Microbiol 32:323–30. Pinner RW, Spellman PA, Stephens DS (1991) Evidence for functionally distinct pili expressed by Neisseria meningitidis. Infect Immun 59:3169–75. Virji M, Alexandrescu C, Ferguson DJ et al (1992)Variations in the expression of pili: the effect on adherence of Neisseria meningitidis to human epithelial and endothelial cells. Mol Microbiol 6:1271–9. Merz AJ, So M (2000) Interactions of pathogenic neisseriae with epithelial cell membranes. Annu Rev Cell Dev Biol 16:423–57.

18

N.G. Rouphael and D.S. Stephens

63. Stephens DS, McGee ZA (1981) Attachment of Neisseria meningitidis to human mucosal surfaces: influence of pili and type of receptor cell. J Infect Dis 143:525–32. 64. Proft T, Baker EN (2009) Pili in Gramnegative and Gram-positive bacteria - structure, assembly and their role in disease. Cell Mol Life Sci 66:613–35. 65. Kahler CM, Martin LE, Tzeng YL et al (2001) Polymorphisms in pilin glycosylation Locus of Neisseria meningitidis expressing class II pili. Infect Immun 69:3597–604. 66. Marceau M, Forest K, Beretti JL et al (1998) Consequences of the loss of O-linked glycosylation of meningococcal type IV pilin on piliation and pilus-mediated adhesion. Mol Microbiol 27:705–15. 67. Callaghan MJ, Jolley KA, Maiden MC (2006) Opacity-associated adhesin repertoire in hyperinvasive Neisseria meningitidis. Infect Immun 74:5085–94. 68. Virji M, Watt SM, Barker S et al (1996) The N-domain of the human CD66a adhesion molecule is a target for Opa proteins of Neisseria meningitidis and Neisseria gonorrhoeae. Mol Microbiol 22:929–39. 69. Virji M, Makepeace K, Ferguson DJ et al (1992) Expression of the Opc protein correlates with invasion of epithelial and endothelial cells by Neisseria meningitidis. Mol Microbiol 6:2785–95. 70. Virji M, Evans D, Hadfield A et al (1999) Critical determinants of host receptor targeting by Neisseria meningitidis and Neisseria gonorrhoeae: identification of Opa adhesiotopes on the N-domain of CD66 molecules. Mol Microbiol 34:538–51. 71. Hill DJ, Griffiths NJ, Borodina E et al (2010) Cellular and molecular biology of Neisseria meningitidis colonization and invasive disease. Clin Sci (Lond) 118:547–64. 72. Perkins-Balding D, Ratliff-Griffin M, Stojiljkovic I (2004) Iron transport systems in Neisseria meningitidis. Microbiol Mol Biol Rev 68:154–71. 73. Schryvers AB, Stojiljkovic I (1999) Iron acquisition systems in the pathogenic Neisseria. Mol Microbiol 32:1117–23. 74. Tzeng YL, Stephens DS (2000) Epidemiology and pathogenesis of Neisseria meningitidis. Microbes Infect 2:687–700. 75. Massari P, Ram S, Macleod H et al (2003) The role of porins in neisserial pathogenesis and immunity. Trends Microbiol 11:87–93. 76. Vermont CL, van Dijken HH, Kuipers AJ et al (2003) Cross-reactivity of antibodies against PorA after vaccination with a meningococcal

77. 78.

79.

80. 81.

82.

83.

84.

85.

86.

87.

88.

89.

90.

B outer membrane vesicle vaccine. Infect Immun 71:1650–5. Rappuoli R (2001) Reverse vaccinology, a genome-based approach to vaccine development. Vaccine 19:2688–91. Frasch CE, Zollinger WD, Poolman JT (1985) Serotype antigens of Neisseria meningitidis and a proposed scheme for designation of serotypes. Rev Infect Dis 7:504–10. Slaterus KW (1961) Serological typing of meningococci by means of micro-precipitation. Antonie Van Leeuwenhoek 27:305–15. Branham S (1953) Serological relationships among meningococci. Bact Rev 17:175–88. Vogel U (2010) Molecular epidemiology of meningococci: Application of DNA sequence typing. Int J Med Microbiol 300(7):415–20. Caugant DA, Froholm LO, Bovre K et al (1986) Intercontinental spread of a genetically distinctive complex of clones of Neisseria meningitidis causing epidemic disease. Proc Natl Acad Sci USA 83:4927–31. Bevanger L, Bergh K, Gisnas G et al (1998) Identification of nasopharyngeal carriage of an outbreak strain of Neisseria meningitidis by pulsed-field gel electrophoresis versus phenotypic methods. J Med Microbiol 47:993–8. Weis N, Lind I (1998) Epidemiological markers in Neisseria meningitidis: an estimate of the performance of genotyping vs phenotyping. Scand J Infect Dis 30:69–75. Maiden MC, Bygraves JA, Feil E et al (1998) Multilocus sequence typing: a portable approach to the identification of clones within populations of pathogenic microorganisms. Proc Natl Acad Sci USA 95:3140–5. Mothershed EA, Sacchi CT, Whitney AM et al (2004) Use of real-time PCR to resolve slide agglutination discrepancies in serogroup identification of Neisseria meningitidis. J Clin Microbiol 42:320–8. Yazdankhah SP, Kriz P, Tzanakaki G et al (2004) Distribution of serogroups and genotypes among disease-associated and carried isolates of Neisseria meningitidis from the Czech Republic, Greece, and Norway. J Clin Microbiol 42:5146–53. Wu HM, Harcourt BH, Hatcher CP et al (2009) Emergence of ciprofloxacin-resistant Neisseria meningitidis in North America. N Engl J Med 360:886–92. Baart GJ, Zomer B, de Haan A et al (2007) Modeling Neisseria meningitidis metabolism: from genome to metabolic fluxes. Genome Biol 8:R136. Price ND, Papin JA, Schilling CH et al (2003) Genome-scale microbial in silico models: the

1 Neisseria meningitidis: Biology, Microbiology, and Epidemiology

91.

92.

93. 94.

95.

96.

97.

98.

99.

100.

101.

102.

103.

104.

constraints-based approach. Trends Biotechnol 21:162–9. Papin JA, Price ND, Wiback SJ et al (2003) Metabolic pathways in the post-genome era. Trends Biochem Sci 28:250–8. Jyssum K, Borchgrevink B, Jyssum S (1961) Glucose catabolism in Neisseria meningitidis. 1. Glucose oxidation and intermediate reactions of the Embden-Meyerhof pathway. Acta Pathol Microbiol Scand 53:71–83. Frantz ID (1942) Growth requirements of the meningococcus. J Bacteriol 1942;43:757–61. Chapin (1918) Carbon dioxide in the primary cultivation of the gonococcus. J Infect Dis 19:558–61. Grossowicz N (1945) Growth requirements and metabolism of Neisseria intracellularis. J Bacteriol 50:109–15. Erwin AL, Gotschlich EC (1996) Cloning of a Neisseria meningitidis gene for L-lactate dehydrogenase (L-LDH): evidence for a second meningococcal L-LDH with different regulation. J Bacteriol 178:4807–13. Leighton MP, Kelly DJ, Williamson MP et al (2001) An NMR and enzyme study of the carbon metabolism of Neisseria meningitidis. Microbiology 147:1473–82. Port JL, DeVoe IW, Archibald FS (1984) Sulphur acquisition by Neisseria meningitidis. Can J Microbiol 30:1453–7. Cohn AC, MacNeil JR, Harrison LH et al (2010) Changes in Neisseria meningitidis disease epidemiology in the United States, 1998–2007: implications for prevention of meningococcal disease. Clin Infect Dis 50:184–91. Fischer M, Hedberg K, Cardosi P et al (1997) Tobacco smoke as a risk factor for meningococcal disease. Pediatr Infect Dis J 16:979–83. Jackson LA, Schuchat A, Reeves MW et al (1995) Serogroup C meningococcal outbreaks in the United States. An emerging threat. JAMA 273:383–9. Rosenstein NE, Perkins BA, Stephens DS et al (1999) The changing epidemiology of meningococcal disease in the United States, 1992– 1996. J Infect Dis 180:1894–901. Cartwright K, Noah N, Peltola H (2001) Meningococcal disease in Europe: epidemiology, mortality, and prevention with conjugate vaccines. Report of a European advisory board meeting Vienna, Austria, 6–8 October, 2000. Vaccine 19:4347–56. Trotter CL, Andrews NJ, Kaczmarski EB et al (2004) Effectiveness of meningococcal

105.

106.

107. 108.

109.

110.

111.

112.

113.

114.

115.

116.

117.

118.

19

serogroup C conjugate vaccine 4 years after introduction. Lancet 364:365–7. Maiden MC, Ibarz-Pavon AB, Urwin R et al (2008) Impact of meningococcal serogroup C conjugate vaccines on carriage and herd immunity. J Infect Dis 197:737–43. Trotter CL, Ramsay ME (2007) Vaccination against meningococcal disease in Europe: review and recommendations for the use of conjugate vaccines. FEMS Microbiol Rev 31:101–7. Lepeyssonnie (1963) La méningite cérébrospinale en Afrique. Bull WHO 28:53–114. Molesworth AM, Cuevas LE, Connor SJ et al (2003) Environmental risk and meningitis epidemics in Africa. Emerg Infect Dis 9:1287–93. Greenwood BM, Bradley AK, Wall RA et al (1985) Meningococcal disease and season in sub-Saharan Africa. Lancet 2:829–30. Lewis R, Nathan N, Diarra L et al (2001) Timely detection of meningococcal meningitis epidemics in Africa. Lancet 358:287–93. Artenstein MS, Rust JH Jr., Hunter DH et al (1967) Acute respiratory disease and meningococcal infection in army recruits. JAMA 201:1004–7. Young LS, LaForce FM, Head JJ et al (1972) A simultaneous outbreak of meningococcal and influenza infections. N Engl J Med 287:5–9. Safadi MA, Cintra OA (2010) Epidemiology of meningococcal disease in Latin America: current situation and opportunities for prevention. Neurol Res32:263–71. Wang JF, Caugant DA, Li X et al (1992) Clonal and antigenic analysis of serogroup A Neisseria meningitidis with particular reference to epidemiological features of epidemic meningitis in the People’s Republic of China. Infect Immun 60:5267–82. Hart CA, Cuevas LE (1997) Meningococcal disease in Africa. Ann Trop Med Parasitol 91:777–85. Wyle FA, Artenstein MS, Brandt BL et al (1972) Immunologic response of man to group B meningococcal polysaccharide vaccines. J Infect Dis 126:514–21. Oster P, Lennon D, O’Hallahan J et al (2005) MeNZB: a safe and highly immunogenic tailor-made vaccine against the New Zealand Neisseria meningitidis serogroup B disease epidemic strain. Vaccine 23:2191–6. Rodriguez AP, Dickinson F, Baly A et al (1999) The epidemiological impact of antimeningococcal B vaccination in Cuba. Mem Inst Oswaldo Cruz 94:433–40.

20

N.G. Rouphael and D.S. Stephens

119. Galloway Y, Stehr-Green P, McNicholas A et al (2009) Use of an observational cohort study to estimate the effectiveness of the New Zealand group B meningococcal vaccine in children aged under 5 years. Int J Epidemiol 38:413–8. 120. Dull PM, Abdelwahab J, Sacchi CT et al (2005) Neisseria meningitidis serogroup W-135 carriage among US travelers to the 2001 Hajj. J Infect Dis 191:33–9. 121. Lingappa JR, Al-Rabeah AM, Hajjeh R et al (2003) Serogroup W-135 meningococcal disease during the Hajj, 2000. Emerg Infect Dis 9:665–71. 122. Wilder-Smith A, Goh KT, Barkham T et al (2003) Hajj-associated outbreak strain of Neisseria meningitidis serogroup W135: estimates of the attack rate in a defined population and the risk of invasive disease developing in carriers. Clin Infect Dis 36:679–83. 123. Hahne SJ, Gray SJ, Jean F et al (2002) W135 meningococcal disease in England and Wales associated with Hajj 2000 and 2001. Lancet 359:582–3. 124. Efron AM, Sorhouet C, Salcedo C et al (2009) W135 invasive meningococcal strains spreading in South America: significant increase in incidence rate in Argentina. J Clin Microbiol 47:1979–80. 125. Koumare B, Ouedraogo-Traore R, Sanou I et al (2007) The first large epidemic of meningococcal disease caused by serogroup W135, Burkina Faso, 2002. Vaccine 25 Suppl 1:A37–41. 126. Traore Y, Njanpop-Lafourcade BM, Adjogble KL et al (2006) The rise and fall of epidemic Neisseria meningitidis serogroup W135 meningitis in Burkina Faso, 2002-2005. Clin Infect Dis 43:817–22.

127. Decosas J, Koama JB (2002) Chronicle of an outbreak foretold: meningococcal meningitis W135 in Burkina Faso. Lancet Infect Dis 2:763–5. 128. Ouedraogo-Traore R, Hoiby EA, Sanou I et al (2002) Molecular characteristics of Neisseria meningitidis strains isolated in Burkina Faso in 2001. Scand J Infect Dis 34:804–7. 129. Materu S, Cox HS, Isaakidis P et al (2007) Serogroup X in meningococcal disease, Western Kenya. Emerg Infect Dis 13:944–5. 130. Boisier P, Nicolas P, Djibo S et al (2007) Meningococcal meningitis: unprecedented incidence of serogroup X-related cases in 2006 in Niger. Clin Infect Dis 44:657–63. 131. Gagneux SP, Hodgson A, Smith TA et al (2002) Prospective study of a serogroup X Neisseria meningitidis outbreak in northern Ghana. J Infect Dis 185:618–26. 132. Harrison LH, Dwyer DM, Maples CT (1999) Risk of meningococcal infection in college students. JAMA 281:1906–10. 133. Kaplan SL, Schutze GE, Leake JA et al (2006) Multicenter surveillance of invasive meningococcal infections in children. Pediatrics 118:e979–84. 134. Shepard CW, Rosenstein NE, Fischer M (2003) Neonatal meningococcal disease in the United States, 1990 to 1999. Pediatr Infect Dis J 22:418–22. 135. Caugant DA (2008) Genetics and evolution of Neisseria meningitidis: importance for the epidemiology of meningococcal disease. Infect Genet Evol 8:558–65. 136. Caugant DA, Maiden MCJ (2009) Meningococcal carriage and disease – population biology and evolution Vaccine 27:B64–B70.

Chapter 2 Classification and Pathogenesis of Meningococcal Infections Petter Brandtzaeg and Marcel van Deuren Abstract The clinical symptoms induced by Neisseria meningitidis reflect compartmentalized intravascular and intracranial bacterial growth and inflammation. In this chapter, we describe a classification system for meningococcal disease based on the nature of the clinical symptoms. Meningococci invade the subarachnoid space and cause meningitis in as many as 50–70% of patients. The bacteremic phase is moderate in patients with meningitis and mild systemic meningococcemia but graded high in patients with septic shock. Three landmark studies using this classification system and comprising 862 patients showed that 37–49% developed meningitis without shock, 10–18% shock without meningitis, 7–12% shock and meningitis, and 18–33% had mild meningococcemia without shock or meningitis. N. meningitidis lipopolysaccharide (LPS) is the principal trigger of the innate immune system via activation of the Toll-like receptor 4-MD2 cell surface receptor complex on myeloid and nonmyeloid human cells. The intracellular signals are conveyed via MyD88-dependent and -independent pathways altering the expression of >4,600 genes in target cells such as monocytes. However, non-LPS molecules contribute to inflammation, but 10–100-fold higher concentrations are required to reach the same responses as induced by LPS. Activation of the complement and coagulation systems is related to the bacterial load in the circulation and contributes to the development of shock, organ dysfunction, thrombus formation, bleeding, and long-term complications in patients. Despite rapid intervention and advances in patient intensive care, why as many as 30% of patients with systemic meningococcal disease develop massive meningococcemia leading to shock and death is still not understood. Key words: Meningococcal meningitis, Septicemia, Classification, Lipopolysaccharide, Shock

1. Introduction Neisseria meningitidis is an obligate human Gram-negative diplococcus residing asymptomatically in the upper respiratory tract (1). The highest carriage rate is found among adolescents and young adults representing the main reservoir of the bacterium. Few young children carry meningococci (1). Most carried strains never cause

Myron Christodoulides (ed.), Neisseria meningitidis: Advanced Methods and Protocols, Methods in Molecular Biology, vol. 799, DOI 10.1007/978-1-61779-346-2_2, © Springer Science+Business Media, LLC 2012

21

22

P. Brandtzaeg and M. van Deuren

invasive disease. A limited number of meningococci belonging to specific clones or clonal complexes with serogroups A, B, C, Y, and W135 cause >95% of systemic meningococcal disease (SMD) (1). Encapsulated meningococci expressing pili, opacity proteins, and other subcapsular adhesion molecules are transferred by droplets or by direct contact (kissing) from an asymptomatic carrier to a nonimmune person. After attachment to nonciliated columnar epithelial cells in the nasopharynx or the epithelium covering the tonsils, meningococci adapt and start to proliferate. Type 4 pili and various outer membrane proteins including opacity proteins undergo phase variation (2). Studies in vitro suggest that N. meningitidis may transverse the epithelial cells through “parasite directed endocytosis” (3). It is assumed that meningococci enter the blood stream through the capillaries and small veins in the underlying submucosal tissue. Survival and growth of encapsulated N. meningitidis in the circulation is a fundamental requirement for developing SMD. Clinical disease usually develops within a week following breach of the mucosal barrier. A century ago, before any effective treatment existed, 70–90% of the patients died during the natural course of infection (4). After the introduction of antimeningococcal serum therapy in 1905, the case fatality rate (CFR) declined to approximately 40% (4). Treatment with sulfonamides in 1937 and with penicillin in 1945 resulted in a further decline of the CFR to approximately 10%. In the last decade, the CFR has ranged from 7 to 11% in Europe and the USA for sporadic cases, increasing to above 20% in outbreak situations (5–7). Two characteristics make N. meningitidis unique among human pathogens: (1) The propensity to invade the meninges and (2) the ability to proliferate rapidly in the blood leading to shock and multiple organ failure in as many as 30% of the patients contracting serogroup B and C strains (6, 8, 9). Meningococci are known among physicians, epidemiologists, and lay people as the bacterium causing outbreaks of meningitis. In industrialized countries, physicians should rightly fear N. meningitidis as the bacterium that may cause overwhelming septicemia, shock, and death. Meningococci may kill previously healthy children or adults within 12–24 h (6, 10, 11). Septic shock and multiple organ failure is the direct cause of death in nine out of ten patients with lethal meningococcal infections in Western countries (Table 1) (6, 8–11). A minority dies of meningitis leading to brain edema, herniation, and arrest of brain circulation (6, 9–11). In Third World countries, particularly in sub-Saharan Africa, N. meningitidis serogroup A still causes large-scale outbreaks involving tens of thousands of people. Meningitis without shock appears to be the dominant clinical presentation (12). A lack of bactericidal antibodies is the single most important predisposing factor for developing SMD (11, 13, 14). Defects in

1983

1987

2008

(8)

(9)

(6)

541

206

115

n

10

17

18

16

29

52

CFR (%)

20

13

15

Shock + meningitis (%)

11

7

12

CFR (%)

49

37

49

Meningitis without shock (%)

1

1

0

CFR (%)

21

33

18

Mild meningococcemia (%)

5

0

0

CFR (%)

n denotes the total number of patients included in the study. CFR denotes case fatality rate of the previous column (6, 8, 9). The clinical presentations are defined as follows: Shock without meningitis: (1) severe septic shock lasting for 24 h or until death requiring fluid and pressor drug therapy and (2) 24 h requiring fluid and pressor drug therapy Shock and meningitis: (1) severe septic shock lasting for 24 h or until death requiring fluid and pressor drug therapy and (2) t100 × 106/L leukocytes (or if the results of CSF are not obtainable, signs of distinct meningism) Meningococcemia without shock or meningitis (mild SMD): Meningococcemia detected by blood culture, PCR or by other methods without (1) severe septic shock requiring treatment for at least 24 h and (2) 97% is required to ensure that there is minimal lymphocyte contamination.

232

H.E. Jones et al.

6. In order to see DC morphology (Fig. 1) it is often easier to immobilize the DC using fibronectin. Sterile plastic 24 well plates are coated with 50 Mg/mL of fibronectin in PBS for 24 h at 37°C. DCs (1 mL, 5 × 106cell/mL) are allowed to adhere to the well for 30 min. 7. It is essential that the cells are kept on ice or at 4°C to prevent internalization of antibodies by Fc receptors expressed on MDDC. It is also important to include isotype controls for each monoclonal antibody to assess Fc receptor-mediated antibody uptake. 8. Cells were analyzed using a FACScalibur flow cytometer with a single argon laser which allows the detection of three fluorophores simultaneously. Typical fluorophore combinations include FITC, PE, and PerCP. DCs are first separated by forward and side scatter where they form a discrete population and this forms the collection gate. At least 5,000 events within this gate are collected for analysis. 9. Although we have focused in this chapter specifically on N. meningitidis, the protocols can also be broadly applicable to infection of DC with other bacteria or bacterial components. 10. For N. meningitidis H44/76, an OD of 1.0 at L600 nm is equal to 1 × 109 bacteria/mL as determined by serial dilution and plating. 11. If live bacteria are being used in a study, it is important to confirm that the bacteria remain viable following FITC labeling. An alternative approach used by us and others is to use bacteria transfected with GFP. 12. The use of fixed bacteria can be useful for a number of reasons: for example, the bacterial concentration does not alter throughout the experiment and using fixed bacteria may be necessary for certain organisms that require containment facilities of level 3 or more. During a natural infection the host is likely to be exposed to both and live and dead organisms, so the response to dead bacteria does have some relevance during a natural infection. Responses of live and killed bacteria can be different, which is the case for N. meningitidis (12), so ideally both states should be compared. 13. DCs should be treated gently and with minimal handling as this can initiate activation. Cells should also be phenotyped to confirm they are immature MDDC as described in Subheading 3.2. 14. It is important to include appropriate positive and negative controls at this stage. Stimulation for 24 h with TNF-A (50 ng/ mL) can be used as a positive control for MDDC maturation and activation. For a negative control, the diluent used for bacterial suspension is appropriate.

14

Human Dendritic Cell Culture and Bacterial Infection

233

15. When using live bacteria it is often necessary to use a range of multiplicities of infection as bacteria can grow during MDDC infection. For example, in 6 h, N. meningitidis numbers can increase tenfold and this should be taken into consideration when comparing live and killed bacteria. 16. The concentration and the time of addition of brefeldin A was optimized for MDDC and in our hands was nontoxic to MDDC stimulated with N. meningitidis. The concentration and time of addition of brefeldin A must be optimized for different stimuli. 17. The concentration of antibody is important. All antibodies should be titrated for optimal staining conditions. If nonspecific binding of an antibody is a problem, then reduce the antibody concentration or the incubation time. 18. We usually use human cytokine ELISA kits from eBioscience, but other manufacturer’s reagents are also suitable for use in this instance. 19. In phagocytosis experiments using flow cytometry, trypan blue staining should be used to assess the proportion of extracellular bacteria compared to intracellular bacteria. 20. It is important to take aliquots at different time points as cells from different donors vary in their ability to phagocytose. It is also worth noting that poor phagocytosis can occur as a result of the greater presence of mature MDDC at the start of the experiment, compared to immature MDDC. 21. Bacterial internalization can be blocked by using an inhibitor of actin polymerization such as cytochalasin D. Add the inhibitor 30 min prior to co-culture with bacteria. In our studies, 1 Mg/ mL of cytochalasin D is optimal to prevent phagocytosis as shown by flow cytometry and confocal analysis. At this concentration, other DC functions such as maturation and cytokine production (other than IL-10 and IL-12) are unaffected. 22. Internalized and surface-bound bacteria are differentiated using this staining method. Surface bound bacteria are FITC (green: emission L520 nm) positive and are also counter-stained with Alexa Fluor 568 specific antibody to P1.7 PorA (red: emission L603 nm). Therefore, extracellular bacteria appear yellow (red and green overlay) whereas intracellular appear green only. DCs are visualized using To-Pro3 which stains the nucleus blue (emission L661 nm). 23. In order to identify internalized bacteria, at least ten optical sections (0.2–0.5 Mm) spanning the entire DC were visualized by Leica confocal imaging software. 24. CFSE diffuses into the T cell, where its acetate group is cleaved by intracellular esterases found in the cytoplasm. This cleavage

234

H.E. Jones et al.

yields a highly fluorescent protein (green) that remains in the cell. Cell division results in a reduction of CFSE fluorescence intensity. 25. Before continuing with the T cell/MDDC co-culture it is good practice to confirm that the CFSE labeling has worked by checking the cells for CFSE incorporation by flow cytometry. 26. T cell cytokines of interest include IL-2 (T cell proliferation inducing cytokine), IFNG (Th1), IL-4 (Th2), and IL-17 (Th17). 27. PMA and ionomycin are used to re-stimulate T cells. PMA is a phorbol ester that activates protein kinase C (PKC) which is important for many cellular processes. Phorbol esters activate PKC because they resemble diacylglycerol. Ionomycin, an ionophore, is used in conjunction with PMA because PKC requires the presence of calcium ions to function. 28. Washing MDDC at this stage is a necessary step to remove excess antigen. If live bacteria have been used to stimulate the cells, it may be important to treat the MDDC with antibiotics, especially as MDDC/T cell co-cultures can last up to 14 days. 29. It is difficult to stimulate naïve T cell responses to study T cell polarization, as the frequency of antigen-specific T cells is very low. This can be overcome by using either an allogeneic (different MDDC and T cell donor) MDDC/T cell co-culture or an autologous MDDC/T cell co-culture, followed by SEB stimulation to further enhance stimulation.

Acknowledgements The authors would like to thank the Meningitis Research Foundation for their financial support. The authors would also like to thank Professor Robin Callard, Dr Heli Uronen-Hansson, and Dr Jenny Allen for their roles in the development of these assays in the Infectious Diseases and Microbiology and Immunobiology Units at the Institute of Child Health, London, UK. References 1. Banchereau J, Briere F, Caux C et al (2000) Immunobiology of dendritic cells. Annu Rev Immunol 18: 767–811. 2. Takeuchi O, Akira A (2010) Pattern recognition receptors and inflammation. Cell 140: 805–20. 3. Murphy K M, Stockinger B (2010) Effector T cell plasticity: flexibility in the face of changing circumstances. Nature Immunol 11: 674–680. 4. Coquerelle C, Mosser M (2010) DC subsets in positive and negative regulation of immunity. Immunol Rev 234: 317–34.

5. Sallusto F, Lanzavecchia A (1994) Efficient presentation of soluble antigen by cultured human dendritic cells is maintained by granulocyte/macrophage colony-stimulating factor plus interleukin 4 and down-regulated by tumor necrosis factor-alpha. J Exp Med 179: 1109–1118. 6. Caux C, Dezutter-Dambuyant C, Schmitt D et al (1992) GM-CSF and TNF-alpha cooperate in the generation of dendritic Langerhans cells. Nature 360: 258 –261.

14

Human Dendritic Cell Culture and Bacterial Infection

7. Bernard H, Disis ML, Heimfeld S et al (1995) Generation of immunostimulatory dendritic cells from human CD34+ hematopoietic progenitor cells of the bone marrow. Cancer Res 55: 1099–1104. 8. Paczesny S, Li Y, Li N et al (2007) Effcient generation of CD34+ progenitor-derived dendritic cells from G-CSF-mobilized peripheral mononuclear cells does not require hematopoietic stem cell enrichment. J Leuk Biol 81: 957–67. 9. Ueno H, Schmitt H, Klechevsky E et al (2010) Harnessing human dendritic cell subsets for medicine. Immunol Rev 234: 199–212. 10. Connolly NC, Whiteside TL, Wilson C et al (2008) Therapeutic immunization with human immunodeficiency virus type 1 (HIV-1) peptideloaded dendritic cell is safe and induces immunogenicity in HIV-1 infected individuals. Clin Vac Immunol 15: 284–292. 11. Villcock A, Schmitt C, Schielke S et al (2008) Recognition via the class A scavenger receptor modulates cytokines secretion by human dendritic cells after contact with Neisseria meningitidis. Microbes Infect 10: 10–11. 12. Jones H E, Uronen-Hansson H, Callard RE et al (2008) The differential response of human dendritic cells to live and killed Neisseria meningitidis. Cell Microbiol 9: 2856–2869. 13. Steeghs L, van Vliet SJ, Uronen-Hansson H et al (2006) Neisseria meningitidis expressing lgtB lipopolysacccharide targets DC-SIGN and modulates dendritic cell function. Cell Microbiol 8: 316–25. 14. Kurzai O, Schmitt C, Claus H et al (2005) Carbohydrate composition of meningococcal lipopolysaccharide modulates the interaction of

235

Neisseria meningitidis with human dendritic cells. Cell Microbiol 7: 1319–1334. 15. Al Bader T, Jolley KA, Humphries HE et al (2004) Activation of human dendritic cells by the PorA protein of Neisseria meningitidis. Cell Microbiol 6: 651–662. 16. Uronen-Hansson H, Steeghs L, Allen J et al (2004) Human dendritic cell activation by Neisseria meningitidis: phagocytosis depends on expression of lipooligosaccharide (LOS) by the bacteria and is required for optimal cytokine production. Cell Microbiol 6: 625–637. 17. Al Bader T, Christodoulides M, Heckels JE et al (2003) Activation of human dendritic cells is modulated by components of the outer membranes of Neisseria meningitidis. Infect Immun 71: 5590–5597. 18. Unkmeir A, Kammerer U, Stade A et al (2002) Lipooligosaccharide and polysaccharide capsule: virulence factors of Neisseria meningitidis that determine meningococcal interaction with human dendritic cells. Infect Immun 70: 2454–2462. 19. Kolb-Maurer A, Unkmeir A, Kammerer U et al (2001) Interaction of Neisseria meningitidis with human dendritic cells. Infect. Immun. 69, 6912–6922. 20. Dixon GL, Newton PJ, Chain BM et al (2001) Dendritic cell activation and cytokine production induced by group B Neisseria meningitidis: interleukin-12 production depends on lipopolysaccharide expression in intact bacteria. Infect Immun 69: 4351–4357. 21. de Jong EC, Vieira PJ, Kalinski P et al (2002) Microbial compounds selectively induce Th1 cell-promoting or Th2 cell-promoting dendritic cells in vitro with diverse Th cell-polarising signals. J Immunol 168: 1704–1709.

wwwwwwwww

Chapter 15 Hydrogen–Deuterium Exchange Coupled to Mass Spectrometry to Investigate Ligand–Receptor Interactions Jessmi M.L. Ling, Leslie Silva, David C. Schriemer, and Anthony B. Schryvers Abstract A method for exploring protein–protein interactions using hydrogen/deuterium exchange coupled to mass spectrometry is described. The method monitors the exchange of backbone (amide) hydrogens in solutions of deuterated water that primarily occur on portions of the protein exposed to solvent. In the presence of a protein binding partner, regions that experience reduced exchange are either part of the protein–protein interaction interface or undergo conformational changes to reduce accessibility to solvent. This method has the advantage of being used under physiological conditions with unmodified proteins. In this chapter, we describe an approach suitable for probing interactions among relatively large proteins using conventional mass spectrometry systems. The interaction between human transferrin and the Neisseria meningitidis receptor protein, transferrin binding protein B, provides a challenging system as an example. Key words: Hydrogen/deuterium exchange, Mass spectrometry, Human transferrin, Transferrin binding protein B, Neisseria meningitidis

1. Introduction Hydrogen/deuterium exchange (H/DX) involves the exposure of protein to a solution containing deuterated water (D2O) such that there is measurable exchange on accessible, labile hydrogens. The exchange rates for hydrogen bonded to carbon are effectively zero, while hydrogen in the side-chains exchanges too rapidly. The hydrogen of backbone amides exchange at a rate that is detectable in H/DX experiments (1). Only residues that are accessible to solvent, such as those on the surface of the protein or in solvent filled channels, will be subject to H/DX in the time course of an H/DX experiment. Proteolytic digestion of a protein exposed to

Myron Christodoulides (ed.), Neisseria meningitidis: Advanced Methods and Protocols, Methods in Molecular Biology, vol. 799, DOI 10.1007/978-1-61779-346-2_15, © Springer Science+Business Media, LLC 2012

237

238

J.M.L. Ling et al.

H/DX followed by separation and analysis of the peptides by mass spectrometry allows identification of deuterated peptides when compared to analysis of peptides from the protein not exposed to D2O. Thus, H/DX coupled to mass spectrometry (H/DX-MS) identifies peptides that are solvent exposed and is capable of monitoring changes in solvent exposure upon perturbations to the system. This feature is especially useful in analyzing protein–protein or protein–ligand interactions (2). Comparative H/DX-MS analysis of the protein in solution, with or without its binding partner, can result in identification of the sites of interaction. The technical challenges associated with H/DX-MS relate to rapid generation and separation of peptides after H/DX in order to minimize the degree of back-exchange, exchanging hydrogen for deuterium in the absence of excess D2O. These steps are normally carried out at low temperature and pH, thus providing a challenge for analysis by LC/MS. Conventional experiments in “bottom-up” protein MS benefit from long digestion periods (overnight) and extended chromatographic separation times (>60 min). In order to retain sufficient deuterium for analysis, digestion times are usually less than 5 min in H/DX-MS experiments, with chromatography complete in 10 min. This results in very complex sets of peptides entering the mass spectrometer at any given time. Large protein complexes can easily “overwhelm” conventional LC/MS systems, leading to poor sequence coverage and a reduced ability to localize binding sites. The approach that was used for dealing with the large size of the partner proteins, human transferrin (hTf) and transferrin binding protein B (TbpB) from Neisseria meningitidis in this example, was to provide a rapid means of separating the proteins after H/DX. This was accomplished by engineering a site for enzymatic biotinylation on the N-terminus of one partner protein (TbpB) so that it could be rapidly separated from the non-tagged partner protein (hTf) under the low pH and temperature of the quenching conditions. Recombinant proteins that are expressed in the Escherichia coli cytoplasm such as TbpB will be endogenously biotinylated, whereas exported or secreted proteins can be biotinylated by recombinant BirA (3). Two additional challenges faced with the H/DX-MS analysis of the hTf used in this study are its resistance to protease due to the high number of disulfide bridges present and the presence of oligosaccharides. These were dealt with by optimizing the composition of the protease digestion buffer and enzymatic removal of the oligosaccharide chains that are described in the methods below. In spite of the challenges, the regions of hTf involved in binding meningococcal TbpB were successfully identified (4) and one might expect higher resolution with proteins that are more susceptible to proteolytic cleavage. Improved resolution of the binding interface and maps of conformational changes may be achieved with enhancements in

15

Hydrogen–Deuterium Exchange Coupled to Mass Spectrometry…

239

LC/MS instrumentation and methods, but the relatively simple approach outlined in this chapter should be suitable for investigators with access to a standard mass spectrometry laboratory.

2. Materials Unless otherwise stated, chemicals are purchased from SigmaAldrich. 2.1. Bacterial Strains, Culture Conditions and Expression of Recombinant TbpB

1. E. coli Top10 carrying a plasmid expressing full-length tbpB from N. meningitidis strain B16B6 (4). E. coli strain C41(DE3) for expression of tbpB (see Note 1). Bacterial strains are stored in 10% (v/v) glycerol in proteose peptone broth at −80°C. 2. GenElute™ Plasmid Miniprep Kit (Sigma-Aldrich). 3. Luria–Bertani (LB) base medium (Invitrogen): 10 g of peptone, 5 g of yeast extract, and 5 g of sodium chloride per liter of distilled water (dH2O). Sterilize using an autoclave at 121°C for 15 min with 2.68 kg/cm2 pressure. 4. LB agar medium (Invitrogen) contains LB base and 15 g per liter of agar. Sterilize using an autoclave at 121°C for 15 min with 2.68 kg/cm2 pressure. 5. LB medium containing 100 Mg/mL of ampicillin: add 1 mL of 1,000× ampicillin stock to 1 L of medium. 6. Recovery medium: LB medium containing 20 mM of glucose. 7. 1,000× ampicillin stock (100 mg/mL): dissolve ampicillin in dH2O and sterilize through a 0.2 Mm pore filter. Store 1 mL aliquots at −20°C until use. 8. Transformation buffer: 100 mM CaCl2 in dH2O. Sterilize using an autoclave at 121°C, for 15 min with 2.68 kg/cm2 pressure and store the solution at 4°C. 9. Water bath set at 42°C. 10. Cell lysis buffer: 50 mM Tris–HCl, pH 8.0 containing 50 mM NaCl. Store at 4°C. 11. Centrifuge with refrigeration capability.

2.2. Enzymatic Deglycosylation of Human Serum Transferrin

1. Deglycosylating enzymes: Peptide-N-glycosidase (PNGase), and endoglycosidases (Endo-) F1, F2, and F3 (4–7). 2. Deglycosylation buffer 1: 100 mM of sodium phosphate (Na2PO4), pH 8.5. 3. Deglycosylation buffer 2: 100 mM of sodium acetate (C2H3NaO2), pH 4.5.

240

J.M.L. Ling et al.

4. Concanavalin A (ConA) sepharose (GE Healthcare). 5. ConA equilibration buffer: 50 mM sodium acetate, pH 6.9 containing 1 mM CaCl2, 1 mM MgCl2, and 1 mM MnCl2. 6. Transferrin storage buffer: 50 mM Tris–HCl, pH 8.0 containing 1 M NaCl. 7. Human serum transferrin (1 mg/mL) in deglycosylation buffer 1. 8. 0.1 N NaOH. 9. Poly-Prep® chromatography columns, 2 mL bed volume, 0.8 × 4 cm (Bio-Rad). 10. Amicon Ultra-15 centrifugal filter device with Ultracel-10 membrane (Millipore). 11. Centrifuge with refrigeration feature that can accommodate 50 mL capacity tubes. A swinging bucket rotor allows more efficient application of the Amicon Ultra-15 centrifugal filter device. 2.3. Preparation of Immobilized TbpB-Transferrin Complex

1. Cell lysis buffer: 50 mM Tris–HCl, pH 8.0 containing 50 mM NaCl. Store at 4°C. 2. High salt wash buffer: 50 mM Tris–HCl, pH 8.0 containing 1 M NaCl. Store at 4°C. 3. Equilibration buffer: 10 mM Tris–HCl, pH 8.0, stored at 4°C. 4. Enzymatically deglycosylated human serum transferrin (40 mg/mL) prepared in cell lysis buffer and stored at 4°C. 5. Streptavidin-agarose (Invitrogen). 6. French pressure cell press (Thermo Spectronic). 7. Centrifuge with refrigeration feature, and capable of higher speeds to pellet cell debris. A suitable benchtop micro-centrifuge for volumes less than 2 mL.

2.4. Peptic Digestion and Generation of Peptide Identification List for Human Serum Transferrin

1. Immobilized pepsin (Thermo Fisher Scientific). 2. Transferrin dilution buffer: 10 mM Tris–HCl, pH 8.0. 3. Pepsin activation buffer: 0.1 M Glycine, pH 2.3 containing 250 mM tris(2-carboxyethyl)phosphine (TCEP)-HCl, and 30% (v/v) methanol. Prepare fresh buffer and chill on ice. 4. A micro-centrifuge. 5. Liquid chromatography (LC) columns. For example, C18 (Magic 200 Å, Michrom BioResources) beads packed in-house in fused silica capillaries (6.5 cm, 360 Mm external diameter, 200 Mm internal diameter). Columns of similar dimensions may be purchased commercially. 6. LC mobile phase A: 3% (v/v) acetonitrile, 0.05% (v/v) trifluoroacetic acid (TFA). 7. LC mobile phase B: 90% (v/v) acetonitrile, 0.05% (v/v) TFA.

15

Hydrogen–Deuterium Exchange Coupled to Mass Spectrometry…

241

8. A quadrupole time-of-flight mass spectrometer: for example, a QSTAR® Pulsar i quadrupole time-of-flight (QqTOF) mass spectrometer (Applied Biosystems), or later model. 9. An liquid chromatography (LC) system capable of delivering reproducible, low flow rates: for example, an Exigent 1D-LC with a loading pump, where gradients can be delivered at