Legionnaires' disease is a severe form of pneumonia that can occur in epidemics of several hundred cases. This atypical pneumonia is characterized by a high mortality rate and affects primarily elderly and immuno-compromised individuals. An in-depth understanding of the ecology and virulence of Legionella spp. will contribute to an efficient and sustainable elimination of the bacteria from anthropogenic water systems and might lead to the development of novel therapeutics. The many different methods described in Legionella: Methods and Protocols are all specifically adapted and developed for the study of distinct features of L. pneumophila, and have already led to exciting discoveries. Most of these techniques can also be applied to the study of other bacterial pathogens, in particular intracellular pathogens like Shigella, Salmonella or Listeria. . Written in the successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible protocols, and notes on troubleshooting and avoiding known pitfalls.
Authoritative and easily accessible, Legionella: Methods and Protocols will not only be useful for research groups studying Legionella, but also for a broader scientific community studying the epidemiology, typing, physiology, pathogenesis, immunity, genetics and evolution of other bacterial pathogens
Table of contents Part 1. Identification and Typing of Legionella in the Environment and During Disease
1. Identification of Legionella in the Environment

Natalia A. Kozak, Claressa E. Lucas, and Jonas M. Winchell
2. Identification of Legionella in Clinical Samples

Sophe Jarraud, Ghislaine Descours, Christophe Ginevra, Gerard Lina, and Jerome Etienne
3. Sequence-Based Identification of Legionella

odney M. Ratcliff
4. Investigation of Outbreaks - Epidemiology

Carol Joseph
5. Outbreak Investigations and Identification of Legionella in Contaminated Water

Susanne Lee and John Lee
6. Typing Methods for Legionella

Christian Lück, Norman K. Fry, Jürgen H. Helbig, Sophie Jarraud, and Timothy G. Harrison

art 2. Physiology and genetics of Legionella
7. Culture, Media and Handling of Legionella

Christa H. Chatfield and Nicholas P. Cianciotto
8. The Intracellular Metabolism of Legionella by Isotopologue Profiling

Klaus Heuner and Wolfgang Eisenreich

9. Induction of Competence for Natural Transformation in Legionella pneumophila and Exploitation for Mutant Construction

Carmen Buchrieser and Xavier Charpentier

10. Constructing Unmarked Gene Deletions in Legionella pneumophila

Andrew Bryan, Zachary D. Abbot, and Michele Swanson
11. Natural Biofilm Formation with Legionella pneumophila

Emilie Portier and Yann Héchard
12. Production of Legionella pneumophila Monospecies Biofilms in a Low-Nutrient Concentration Medium

Sophie Pécastaings and Christine Roques
13. Enrichment of Outer Membrane Vesicles Shed by Legionella pneumophila

Jens Jäger and Michael Steinert

Part 3. Cellular microbiology of Legionella
14. Analysis of Legionella Infection by Flow Cytometry

Andre Tiaden, Aline Kessler, and Hubert Hilbi

15. Analysis of Legionella Infection Using RNAi in Drosophila Cells

Dennise A. De Jesús, Tamara J. O'Connor, and Ralph R. Isberg

16. Immunofluorescence Imaging of Legionella Infection In vitro
Stefan S. Weber and Annette Oxenius
17. Electron Microscopy of Legionella and Legionella-Infected Cells
Gary Faulkner and Raphael Garduno
18. Pathogen Vacuole Purification from Legionella-Infected Amoeba and Macrophages

Christine Hoffmann, Ivo Finsel, and Hubert Hilbi

19. Methods for Determining Protein Translocation by the Legionella pneumophila Dot/Icm Type IV Secretion System

Wenhan Zhu and Zhao-Qing Luo

20. Subcellular Localization of Legionella Dot/Icm Effectors

Adam J. Vogrin, Aurelie Mousnier, Gad Frankel, and Elizabeth L. Hartland

art 4. Biochemical assays to study Legionella effectors and enzymes
21. Purification and Characterization of Legionella U-Box-Type E3 ubiquitin ligase

Hiroki Nagai and Tomoko Kubori
22. Legionella Phospholipases Implicated in Infection Determination of Enzymatic Activities

Kathleen Seipel and Antje Flieger

3. Interactions of Legionella Effector Proteins with Host Phosphoinositide Lipids

Stephen Weber, Stephanie Dolinsky, and Hubert Hilbi

24. Characterization of Legionella lipopolysaccharide

Christian Lück and Jürgen Helbig

art 5. Immunity and host response against Legionella

5. The Tetrahymena and Acanthamoeba Model Systems

Sharon G. Berk and Rafael A. Garduño

26. Dictyostelium Host Response to Legionella infection: Strategies and Assays

Salvatore Bozzaro, Barbara Peracino, and Ludwig Eichinger
27. The Caenorhabditis elegans Model of Legionella Infection

Ann Karen C. Brassinga and Costi D. Sifri
28. Infection of Nonphagocytic Host Cells by Legionella

Shandra R. Day, Costi D. Sifri, and Paul S. Hoffman
29. Analyzing Caspase-1 Activation During Legionella pneumophila Infection in Macrophages

Christopher L. Case and Craig R. Roy
30. The Mouse as a Model for Pulmonary Legionella Infection

Juliana I. Hori and Dario Zamboni
31. Assessment of Legionella-Specific Immunity in Mice

Stefan Weber, Nicole Joller, and Annette Oxenius

32. The Guinea Pig Model of Legionnaires' Disease

Paul H. Edelstein

33. Human Susceptibility to Legionnaires Disease

William R. Berrington and Thomas R. Hawn

Part 6. Genomics, transcriptomics and proteomics of Legionella
34. cDNA library Construction for Next Generation Sequencing (NGS) to Determine the Transcriptional Landscape of Legionella pneumophila

Tobias Sahr and Carmen Buchrieser
35. Methods to Study Legionella Transcriptome in vitro and in vivo

Sebastian Faucher and Howard Shuman
36. Co-Immunoprecipitation: Protein-RNA and Protein-DNA Interaction

Tobias Sahr and Carmen Buchrieser
37. Identification of Legionella Effectors Using Bioinformatic Approaches

Gil Segal
Part 5. Immunity and host response against Legionella
25. The Tetrahymena and Acanthamoeba Model Systems

ron G. Berk and Rafael A. Garduño

26. Dictyostelium Host Response to Legionella infection: Strategies and Assays

Salvatore Bozzaro, Barbara Peracino, and Ludwig Eichinger

27. The Caenorhabditis elegans Model of Legionella Infection

Ann Karen C. Brassinga and Costi D. Sifri

28. Infection of Nonphagocytic Host Cells by Legionella

Shandra R. Day, Costi D. Sifri, and Paul S. Hoffman

29. Analyzing Caspase-1 Activation During Legionella pneumophila Infection in Macrophages

Christopher L. Case and Craig R. Roy

30. The Mouse as a Model for Pulmonary Legionella Infection

Juliana I. Hori and Dario Zamboni

31. Assessment of Legionella-Specific Immunity in Mice

Stefan Weber, Nicole Joller, and Annette Oxenius

32. The Guinea Pig Model of Legionnaires' Disease

Paul H. Edelstein


33. Human Susceptibility to Legionnaires Disease

William R. Berrington and Thomas R. Hawn


Part 6. Genomics, transcriptomics and proteomics of Legionella


34. cDNA library Construction for Next Generation Sequencing (NGS) to Determine the Transcriptional Landscape of Legionella pneumophila

Tobias Sahr and Carmen Buchrieser


35. Methods to Study Legionella Transcriptome in vitro and in vivo

Sebastian Faucher and Howard Shuman


36. Co-Immunoprecipitation: Protein-RNA and Protein-DNA Interaction

Tobias Sahr and Carmen Buchrieser



37. Identification of Legionella Effectors Using Bioinformatic Approaches

Gil Segal