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
Rodney 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
Part 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
Part 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
23. 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
Part 5. Immunity and host response against Legionella
25. 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
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