Mycobacteria Protocols

M E T H O D S I N M O L E C U L A R B I O L O G YTM

John M. Walker, SERIES EDITOR

484. Functional Proteomics: Methods and Protocols,edited by Julie D. Thompson, ChristineSchaeffer-Reiss, and Marius Ueffing, 2008

483. Recombinant Proteins From Plants: Methodsand Protocols, edited by Loıc Faye and VeroniqueGomord, 2008

482. Stem Cells in Regenerative Medicine: Methodsand Protocols, edited by Julie Audet and WilliamL. Stanford, 2008

481. Hepatocyte Transplantation: Methods andProtocols, edited by Anil Dhawan and Robin D.Hughes, 2008

480. Macromolecular Drug Delivery: Methods andProtocols, edited by Mattias Belting, 2008

479. Plant Signal Transduction: Methods andProtocols, edited by Thomas Pfannschmidt, 2008

478. Transgenic Wheat, Barley and Oats:Production and Characterization Protocols,edited by Huw D. Jones and Peter R. Shewry, 2008

477. Advanced Protocols in Oxidative Stress I,edited by Donald Armstrong, 2008

476. Redox-Mediated Signal Transduction: Methodsand Protocols, edited by John T. Hancock, 2008

475. Cell Fusion: Overviews and Methods, edited byElizabeth H. Chen, 2008

474. Nanostructure Design: Methods and Protocols,edited by Ehud Gazit and Ruth Nussinov, 2008

473. Clinical Epidemiology: Practice and Methods,edited by Patrick Parfrey and Brendon Barrett,2008

472. Cancer Epidemiology, Volume 2: ModifiableFactors, edited by Mukesh Verma, 2008

471. Cancer Epidemiology, Volume 1: HostSusceptibility Factors, edited by Mukesh Verma,2008

470. Host-Pathogen Interactions: Methods andProtocols, edited by Steffen Rupp and Kai Sohn,2008

469. Wnt Signaling, Volume 2: Pathway Models,edited by Elizabeth Vincan, 2008

468. Wnt Signaling, Volume 1: Pathway Methods andMammalian Models, edited by Elizabeth Vincan,2008

467. Angiogenesis Protocols: Second Edition, editedby Stewart Martin and Cliff Murray, 2008

466. Kidney Research: Experimental Protocols, editedby Tim D. Hewitson and Gavin J. Becker, 2008.

465. Mycobacteria Protocols ,

461. Molecular Embryology: Methods and Protocols,Second Edition, edited by Paul Sharpe and IvorMason, 2008

460. Essential Concepts in Toxicogenomics, edited byDonna L. Mendrick and William B. Mattes, 2008

459. Prion Protein Protocols, edited by Andrew F.Hill, 2008

458. Artificial Neural Networks: Methods andApplications, edited by David S. Livingstone,2008

457. Membrane Trafficking, edited by Ales Vancura,2008

456. Adipose Tissue Protocols, Second Edition, editedby Kaiping Yang, 2008

455. Osteoporosis, edited by Jennifer J. Westendorf,2008

454. SARS- and Other Coronaviruses: LaboratoryProtocols, edited by Dave Cavanagh, 2008

453. Bioinformatics, Volume 2: Structure, Function,and Applications, edited by Jonathan M. Keith,2008

452. Bioinformatics, Volume 1: Data, SequenceAnalysis, and Evolution, edited by JonathanM. Keith, 2008

451. Plant Virology Protocols: From Viral Sequenceto Protein Function, edited by Gary Foster,Elisabeth Johansen, Yiguo Hong, and Peter Nagy,2008

450. Germline Stem Cells, edited by Steven X. Houand Shree Ram Singh, 2008

449. Mesenchymal Stem Cells: Methods andProtocols, edited by Darwin J. Prockop, DouglasG. Phinney, and Bruce A. Brunnell, 2008

448. Pharmacogenomics in Drug Discovery andDevelopment, edited by Qing Yan, 2008.

447. Alcohol: Methods and Protocols, edited byLaura E. Nagy, 2008

446. Post-translational Modifications of Proteins:Tools for Functional Proteomics, Second Edition,edited by Christoph Kannicht, 2008.

445. Autophagosome and Phagosome, edited by VojoDeretic, 2008

444. Prenatal Diagnosis, edited by Sinhue Hahn andLaird G. Jackson, 2008.

443. Molecular Modeling of Proteins, edited byAndreas Kukol, 2008.

442. RNAi: Design and Application, edited by SailenBarik, 2008.

441. Tissue Proteomics: Pathways, Biomarkers, andDrug Discovery, edited by Brian Liu, 2008

440. Exocytosis and Endocytosis, edited by Andrei I.Ivanov, 2008

439. Genomics Protocols, Second Edition, edited byMike Starkey and Ramnanth Elaswarapu, 2008

438. Neural Stem Cells: Methods and Protocols,Second Edition, edited by Leslie P. Weiner, 2008

437. Drug Delivery Systems, edited by Kewal K. Jain,2008

, Second Editionedited by Tanya Parish and Amanda Claire

464. The Nucleus, Volume 2: Physical Properties andImaging Methods, edited by Ronald Hancock,2008

463. The Nucleus, Volume 1: Nuclei and SubnuclearComponents, edited by Ronald Hancock, 2008

462. Lipid Signaling Protocols, edited by BanafsheLarijani, Rudiger Woscholski, and Colin A.Rosser, 2008

Brown, 2008

M E T H O D S I N M O L E C U L A R B I O L O G Y T M

MycobacteriaProtocols

Second Edition

Edited by

Tanya Parish andAmanda Claire Brown

Barts and The LondonQueen Mary’s School of Medicine

and DentistryLondon, UK

EditorsTanya ParishBarts and The LondonQueen Mary’s School of Medicineand Dentistry,

London, UKt.parish@qmul.ac.uk

Amanda Claire BrownBarts and The LondonQueen Mary’s School of Medicineand Dentistry,

London, UKa.c.brown@qmul.ac.uk

Series EditorJohn M. WalkerUniversity of HertfordshireHatfield, Hertfordshire,UK

ISBN: 978-1-58829-889-8 e-ISBN: 978-1-59745-207-6ISSN: 1064-3745 e-ISSN: 1940-6029DOI: 10.1007/978-1-59745-207-6

Library of Congress Control Number: 2008933364

# 2008 Humana Press, a part of Springer ScienceþBusiness Media, LLCAll rights reserved. This workmay not be translated or copied in whole or in part without the writtenpermission of the publisher (Humana Press, c/o Springer Science+Business Media, LLC, 233 SpringStreet, New York, NY 10013, USA), except for brief excerpts in connection with reviews orscholarly analysis. Use in connection with any form of information storage and retrieval,electronic adaptation, computer software, or by similar or dissimilar methodology now known orhereafter developed is forbidden.The use in this publication of trade names, trademarks, service marks, and similar terms, even if theyare not identified as such, is not to be taken as an expression of opinion as to whether or not they aresubject to proprietary rights.

Printed on acid-free paper

9 8 7 6 5 4 3 2 1

springer.com

Preface

Themycobacteria include a number of important human and animal pathogensand pose major problems worldwide in terms of global health and economies.Tuberculosis poses a significant threat to global health by infecting and killingmillions annually. Leprosy has not yet been eradicated, and other infections,such as Buruli ulcer and opportunistic infections associated with immuno-deficiency, are on the rise. For these reasons, the need for methods to studythe biology of the mycobacteria and to improve diagnostic, therapeutic, andpreventative reagents is still a priority.

It has been nearly 10 years since the first edition of Mycobacteria Protocols.The response to the first edition was both surprising and pleasing.Many readerscommented on how the book had helped them with a tricky problem or allowedthem to branch into new areas, and newcomers to the field were able to avoidmany common pitfalls and progress quickly. Mycobacteria are difficult organ-isms to work with, but the availability of the tips and tricks developed by amultitude of scientists over many years has been received very positively.Within my own laboratory, this book has proved invaluable for neophytes.During the time since it was published, research into the mycobacteria hascontinued to expand, and the number of scientists studying these problematicbacteria has increased. New methods have been developed, and older methodshave been refined, making a second edition timely.

In this second edition, we have tried to include a range of methods, from thebasics of subcellular fractionation, strain typing, and determining minimuminhibitory concentrations (MICs) to more advanced methods using specializedgrowth conditions and whole genome, transcriptome, and proteome analyses.Although we cannot include all methods used in the modern research labora-tory, we hope that this edition will provide a useful primer for new mycobac-terial researchers and stimulate new avenues for established scientists.

In developing this edition, we have revised and updated some of the mostused methods from the original version and incorporated a number of whollynew methods, some of which make use of our expanded horizons in the post–genomic age. Several methods are described forMycobacterium tuberculosis, asthis is now the most widely studied species, but can be easily adapted for othermembers of the genus.

v

Updated and revised methods include techniques for subcellular fractiona-tion, notably the isolation of genomic DNA and RNA—techniques that arenow more useful than ever given the availability of whole genome microarrays.Methods for identifying deletions in genomic DNA and for assaying genome-wide gene expression patterns are presented to make use of these fractions.Analysis of the proteome by two-dimensional gel electrophoresis is alsoincluded. These methods will allow the exploitation of the information affordedby the availability of the whole genome sequence for several mycobacterialspecies. Such a concerted sequencing effort has provided a plethora of informa-tion, although this can sometimes be overwhelming. MycoDB is an onlineresource designed to facilitate genomic analyses of Mycobacterium spp. andrelated genera, and a chapter explaining this powerful engine is included.

Mycobacteria have a characteristic lipid-rich cell wall. This outer layer isresponsible for a number of their features, including the acid-fast property usedto identify the genus. Cell wall composition has implications in many otherareas, from immune recognition to insensitivity to antibiotics. A number oftechniques are presented to allow the reader to analyze the composition of thecell wall including the complex lipoglycans, which include the immunomodu-latory molecule lipoarabinomannan. Lipids plays an important role in myco-bacteria, and methods for analyzing lipid biosynthesis and location as wellas metabolism are detailed. The cell wall forms a strong barrier to manyantimicrobials, and the use of transport assays to measure permeability isdiscussed.

Genetic techniques for manipulating the genome of mycobacteria havegreatly improved since the past edition. Introducing recombinant DNA intomycobacteria is not as straightforward as for other bacterial species, so anupdated version of the electroporation chapter is included. Much has happenedin the plasmid field, and an updated review of the most commonly usedvectors is given together with methods for using the only available temperature-sensitive plasmid, and two chapters deal with expression of genes in mycobac-teria using inducible plasmid systems. Homologous recombination has beenused to construct mutants for many years, and two methods are described here.Other methods for creating mutants have also been developed, and phagetransposon mutagenesis is described. The identification of mycobacterialpromoters and analysis of gene expression is a growing area of research, anda chapter that describes the use of two reporter systems, powerful tools forinvestigating transcriptional regulation in response to environmental signals,is included. A great deal of interest in identifying essential genes has arisen,and two techniques are provided for checking the essentiality of a given gene.The first method, CESTET, can rapidly identify essential genes. The secondmethod, gene switching, can also be used to study the role of essential genesfurther.

The mycobacteria include several pathogens, so there is a need forstrain identification in molecular epidemiology and genome variation studies.Methods for typing strains using insertion sequence, IS6110, and the newer

vi Preface

PCR-based method of analyzing repeat units (MIRU/VNTR) are given.The determination of MICs can be technically difficult, so protocols that canbe used with clinical isolates and an Alamar blue–based method that could beapplied to the development of novel inhibitors are included. A method forlooking at molecular drug resistance using a scanning-frame oligonucleotidemicroarray is also included.

We have included a chapter describing the use of a chemostat to growmycobacterial cultures under highly defined conditions, which is useful forstudying environmental responses. One particular environment of interest tomycobacteriologists is the intracellular environment, as several mycobacterialspecies can survive and multiply inside eukaryotic cells. A model for this isprovided in a chapter dealing with survival and gene expression in amoebae.

We hope that this book helps to promote and stimulate further research intothese intriguing, important, and most fractious of bacteria. We would like tothank all the authors who have made this second edition possible and hope thatthese updated and new protocols will continue to serve the mycobacterialresearch community as a useful resource.

Tanya ParishAmanda Claire Brown

Preface vii

Contents

Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v

Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xiii

1. Isolation of Mycobacterium Species Genomic DNA . . . . . . . . . . . . . . . . . 1John T. Belisle, Spencer B. Mahaffeyand Preston J. Hill

2. Isolation of Mycobacterial RNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13Tige R. Rustad, David M. Roberts, Reiling P. Liaoand David R. Sherman

3. Lipoglycans of Mycobacterium tuberculosis: Isolation,Purification, and Characterization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23Libin Shi, Jordi B. Torrelles andDelphi Chatterjee

4. Analyzing Lipid Metabolism: Activation and b-Oxidation

of Fatty Acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47Paul Robert Wheeler

5. Analysis of Lipid Biosynthesis and Location . . . . . . . . . . . . . . . . . . . . . . 61Paul Robert Wheeler

6. Whole Genome Analysis Using Microarrays . . . . . . . . . . . . . . . . . . . . . 83S.J. Waddell, J. Hinds andP.D. Butcher

7. Use of DNA Microarrays to Study Global Patterns

of Gene Expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95Roberta Provvedi, Giorgio Palu andRiccardo Manganelli

ix

8. Two-Dimensional Gel Electrophoresis–Based Proteomics

of Mycobacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111Jens Mattow, Frank Siejak, Kristine Hagens, Julia Kreuzeder,Stefan H.E. Kaufmann and Ulrich E. Schaible

9. Transport Assays and Permeability in Pathogenic Mycobacteria . . . . . 143Marie-Antoinette Laneelle and Mamadou Daffe

10. Continuous Culture of Mycobacteria . . . . . . . . . . . . . . . . . . . . . . . . . 153Joanna Bacon and Kim A. Hatch

11. Measuring Minimum Inhibitory Concentrations in Mycobacteria . . . . 173Frederick A. Sirgel, Ian J.F. Wiid and Paul D. van Helden

12. Rapid Screening of Inhibitors of Mycobacterium tuberculosisGrowth Using Tetrazolium Salts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 187Anita G. Amin, Shiva K. Angala, Delphi Chatterjeeand Dean C. Crick

13. Electroporation of Mycobacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . 203Renan Goude and Tanya Parish

14. Ins and Outs of Mycobacterial Plasmids . . . . . . . . . . . . . . . . . . . . . . . 217Farahnaz Movahedzadeh and Wilbert Bitter

15. The Use of Temperature-Sensitive Plasmids in Mycobacteria . . . . . . . 229Damien Portevin, Wladimir Malaga and Christophe Guilhot

16. Heterologous Expression of Genes in Mycobacteria . . . . . . . . . . . . . . 243James A. Triccas and Anthony A. Ryan

17. Inducible Expression Systems for Mycobacteria . . . . . . . . . . . . . . . . . 255Christopher M. Sassetti

18. Assaying Promoter Activity Using LacZ and GFP as Reporters . . . . 265Paul Carroll and Jade James

19. Construction of Unmarked Deletion Mutants in Mycobacteria . . . . . . 279Houhui Song, Frank Wolschendorf andMichael Niederweis

20. Construction of Targeted Mycobacterial Mutants by Homologous

Recombination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 297Sharon L. Kendall and Rosangela Frita

x Contents

21. Phage Transposon Mutagenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 311M. Sloan Siegrist and Eric J. Rubin

22. Gene Essentiality Testing in Mycobacterium smegmatisUsing Specialized Transduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 325Apoorva Bhatt and William R. Jacobs Jr.

23. Gene Switching and Essentiality Testing . . . . . . . . . . . . . . . . . . . . . . . 337Amanda Claire Brown

24. Insertion Element IS6110-Based Restriction Fragment Length

Polymorphism Genotyping of Mycobacterium tuberculosis . . . . . . . . . 353Robin M. Warren, Paul D. van Heldenand Nicolaas C. Gey van Pittius

25. Typing Mycobacterium tuberculosis Using Variable Number

Tandem Repeat Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 371T.J. Brown, V.N. Nikolayevskyy and F.A. Drobniewski

26. Molecular Detection of Drug-Resistant Mycobacteriumtuberculosis with a Scanning-Frame Oligonucleotide Microarray . . . . 395Dmitriy V. Volokhov, Vladimir E. Chizhikov, Steven Denkinand Ying Zhang

27. MycoDB: An Online Database for Comparative Genomics

of the Mycobacteria and Related Organisms . . . . . . . . . . . . . . . . . . . 419Roy R. Chaudhuri

28. Environmental Amoebae and Mycobacterial Pathogenesis . . . . . . . . . 433Melanie Harriff and Luiz E. Bermudez

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 443

Contents xi

Contributors

Anita G. Amin, MSDepartment of Microbiology, Immunology and Pathology, Colorado StateUniversity, Fort Collins, Colorado, USA

Shiva K. Angala, MSDepartment of Microbiology, Immunology and Pathology, Colorado StateUniversity, Fort Collins, Colorado, USA

Joanna Bacon, PhDTB Research Programme, CEPR, Health Protection Agency, Porton Down,Salisbury, UK

John T. Belisle, PhDDepartment of Microbiology, Immunology and Pathology, Colorado StateUniversity, Fort Collins, Colorado, USA

Luiz E. Bermudez, MDBiomedical Sciences, College of Veterinary Medicine, Oregon State University,Corvallis, Oregon, USA

Apoorva Bhatt, PhDSchool of Biosciences, University of Birmingham, Edgbaston, Birmingham, UK

Wilbert Bitter, PhDMedical Microbiology, VU University Medical Centre, Amsterdam,The Netherlands

Amanda Claire Brown, PhDCentre for Infectious Disease, Institute of Cell and Molecular Science, Bartsand The London, Queen Mary’s School of Medicine and Dentistry,London, UK

xiii

T.J. Brown, PhDHealth Protection Agency Mycobacterium Reference Unit, Bartsand The London, Queen Mary’s School of Medicine and Dentistry,London, UK

P.D. Butcher, PhDDivision of Cellular and Molecular Medicine, St. George’s Universityof London, London, UK

Paul Carroll, PhDCentre for Infectious Disease, Institute of Cell and Molecular Science,Barts and The London, Queen Mary’s School of Medicine and Dentistry,London, UK

Delphi Chatterjee, PhDDepartment of Microbiology, Immunology and Pathology, Colorado StateUniversity, Fort Collins, Colorado, USA

Roy R. Chaudhuri, PhDDepartment of Veterinary Medicine, Cambridge Veterinary School,University of Cambridge, Cambridge, UK

Vladimir E. Chizhikov, PhDCenter for Biologics Evaluation and Research, U.S. Food and DrugAdministration, Rockville, Maryland, USA

Dean C. Crick, PhDDepartment of Microbiology, Immunology and Pathology, Colorado StateUniversity, Fort Collins, Colorado, USA

Mamadou Daffe, PhDDepartment of Molecular Mechanisms of Mycobacterial Infections, Instituteof Pharmacology and Structural Biology, UMR 5089 of the National Centrefor Scientific Research (CNRS) and University Paul Sabatier (Toulouse III),Toulouse France

Steven Denkin, PhDDepartment of Molecular Microbiology and Immunology,Bloomberg School of Public Health, Johns Hopkins University, Baltimore,Maryland, USA

F.A. Drobniewski, PhD, MBBSHealth Protection Agency Mycobacterium Reference Unit, Bartsand The London, Queen Mary’s School of Medicine and Dentistry,London, UK

xiv Contributors

Rosangela Frita, BScDepartment of Pathology and Infectious Diseases, The Royal VeterinaryCollege, London, UK

Nicolaas C. Gey van Pittius, PhDDepartment of Biomedical Sciences, Faculty of Health Sciences, StellenboschUniversity, Cape Town, South Africa

Renan Goude, PhDCentre for Infectious Disease, Institute of Cell and Molecular Science,Barts and The London, Queen Mary’s School of Medicine and Dentistry,London, UK

Christophe Guilhot, PhDCentre National de la Recherche Scientifique and Universite Paul Sabatier,Institut de Pharmacologie et de Biologie Structurale, DepartementMecanismesMoleculaires des Infections Mycobacteriennes, Toulouse, France

Kristine Hagens, TA (Technician)Department of Immunology, Max Planck Institute for Infection Biology,Berlin, Germany

Melanie Harriff, PhDVollum Institute for Biomedical Research, Oregon Health and SciencesUniversity, Portland, Oregon, USA

Kim A. Hatch, MScTB Research Programme, CEPR, Health Protection Agency, Porton Down,Salisbury, UK

Preston J. Hill, BScDepartment of Microbiology, Immunology and Pathology, Colorado StateUniversity, Fort Collins, Colorado, USA

J. Hinds, PhDDivision of Cellular and Molecular Medicine, St. George’s Universityof London, London, UK

William R. Jacobs Jr., PhDDepartment of Microbiology and Immunology, Howard Hughes MedicalInstitute, Albert Einstein College of Medicine, Bronx, New York, USA

Jade James, BScCentre for Infectious Disease, Institute of Cell and Molecular Science, Bartsand The London, Queen Mary’s School of Medicine and Dentistry,London, UK

Contributors xv

Stefan H.E. Kaufmann, Prof., PhDDepartment of Immunology, Max Planck Institute for Infection Biology,Berlin, Germany

Sharon L. Kendall, PhDDepartment of Pathology and Infectious Diseases, The Royal VeterinaryCollege, London, UK

Julia Kreuzeder, PhDDepartment of Immunology, Max Planck Institute for Infection Biology,Berlin, Germany

Marie-Antoinette Laneelle, PhDDepartment of Molecular Mechanisms of Mycobacterial Infections, Institutof Pharmacology and Structural Biology, UMR 5089 of the National Centrefor Scientific Research (CNRS) and University Paul Sabatier (Toulouse III),Toulouse France

Reiling P. Liao, MSSeattle Biomedical Research Institute, Seattle, Washington, USA

Spencer B. Mahaffey, BScDepartment of Microbiology, Immunology and Pathology, Colorado StateUniversity, Fort Collins, Colorado, USA

Wladimir Malaga, PhDCentre National de la Recherche Scientifique and Universite Paul Sabatier,Institut de Pharmacologie et de Biologie Structurale, DepartementMecanismesMoleculaires des Infections Mycobacteriennes, Toulouse, France

Riccardo Manganelli, PhDDepartment of Histology, Microbiology and Medical Biotechnologies,University of Padua, Padua, Italy

Jens Mattow, PhDDepartment of Immunology, Max Planck Institute for Infection Biology,Berlin, Germany

Farahnaz Movahedzadeh, PhDInstitute for Tuberculosis Research, University of Illinois at Chicago (UIC),Chicago, Illinois, USA

Michael Niederweis, PhDDepartment of Microbiology, University of Alabama at Birmingham,Birmingham, Alabama, USA

xvi Contributors

V.N. Nikolayevskyy, PhDHealth Protection Agency Mycobacterium Reference Unit, Bartsand The London, Queen Mary’s School of Medicine and Dentistry,London, UK

Giorgio Palu, MDDepartment of Histology, Microbiology and Medical Biotechnologies,University of Padua, Padua, Italy

Tanya Parish, PhDCentre for Infectious Disease, Institute of Cell and Molecular Science,Barts and The London, Queen Mary’s School of Medicine and Dentistry,London, UK

Damien Portevin, PhDMycobacterial Division Research, National Institute for Medical Research,London, UK

Roberta Provvedi, PhDDepartment of Biology, University of Padua, Padua, Italy

David M. Roberts, PhDSeattle Biomedical Research Institute, Seattle, Washington, USA

Eric J. Rubin, MD, PhDDepartment of Immunology and Infectious Diseases, Harvard School of PublicHealth, Boston, Massachusetts, USA

Tige R. Rustad, PhDSeattle Biomedical Research Institute, Seattle, Washington, USA

Anthony A. Ryan, PhDCentenary Institute of Cancer Medicine and Cell Biology, Universityof Sydney, Sydney, Australia

Christopher M. Sassetti, PhDDepartment of Molecular Genetics and Microbiology, Universityof Massachusetts Medical School, Worcester, Massachusetts, USA

Ulrich E. Schaible, PhDLondon School of Hygiene and Tropical Medicine, London, UK

David R. Sherman, PhDSeattle Biomedical Research Institute, Seattle, Washington, USA

Contributors xvii

Libin Shi, PhDColorado State University, Department of Microbiology, Immunologyand Pathology, Fort Collins, Colorado, USA

M. Sloan Siegrist, PhDDepartment of Immunology and Infectious Diseases, Harvard School of PublicHealth, Boston, Massachusetts, USA

Frank SiejakDepartment of Immunology, Max Planck Institute for Infection Biology,Berlin, Germany

Frederick A. Sirgel, PhDDivision of Molecular Biology and Human Genetics, MRC Centrefor Molecular and Cellular Biology, DST/NRF Centre of Excellencefor Biomedical TB Research, Stellenbosch University, Tygerberg, South Africa

Houhui Song, PhDDepartment of Microbiology, University of Alabama at Birmingham,Birmingham, Alabama, USA

Jordi B. Torrelles, PhDDepartment of Internal Medicine and Division of Infectious Diseases,The Ohio State University, Columbus, Ohio, USA

James A. Triccas, PhDDiscipline of Infectious Diseases and Immunology, University of Sydney,Sydney, Australia

David Paul van Helden, PhDMRC Centre for Molecular and Cellular Biology, DST/NRF Centreof Excellence for Biomedical TB Research, Stellenbosch University, Tygerberg,South Africa

Dmitriy V. Volokhov, PhDCenter for Biologics Evaluation and Research, U.S. Food and DrugAdministration, Rockville, Maryland, USA

S.J. Waddell, PhDDivision of Cellular and Molecular Medicine, St. George’s Universityof London, London, UK

Robin M. Warren, PhDDivision of Molecular Biology and Human Genetics, MRC Centrefor Molecular and Cellular Biology, DST/NRF Centre of Excellencefor Biomedical TB Research, Stellenbosch University, Tygerberg, South Africa

xviii Contributors

Paul Robert Wheeler, PhDTuberculosis Research Group, Veterinary Laboratories Agency (Weybridge),Addlestone, Surrey, UK

Ian J.F. Wiid, PhDDivision of Molecular Biology and Human Genetics, MRC Centrefor Molecular and Cellular Biology, DST/NRF Centre of Excellencefor Biomedical TB Research, Stellenbosch University, Tygerberg, South Africa

Frank Wolschendorf, PhDDepartment of Microbiology, University of Alabama at Birmingham,Birmingham, Alabama, USA

Ying Zhang, MD, PhDDepartment of Molecular Microbiology and Immunology, Bloomberg Schoolof Public Health, Johns Hopkins University, Baltimore, Maryland, USA

Contributors xix