Legionella pp 233-249 | Cite as

Analysis of Legionella Infection by Flow Cytometry

Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 954)

Abstract

Legionella pneumophila infects and replicates in environmental protozoa and metazoan macrophages within a specific vacuole. The infection of phagocytes by L. pneumophila can be assessed by an agar plating assay or by fluorescence microscopy. Here, we describe the analysis of Legionella infection by automated flow cytometry using wild-type and mutant bacteria that constitutively produce the green fluorescent protein (GFP). Advantages of the flow cytometry technique include (1) a software-assisted multiple parameter analysis of Legionella infections in real-time at distinct stages of the infection cycle, (2) the simultaneous and fast acquisition of a high number of data points, and (3) a characterization of the infecting bacteria in parallel with the infected host cells.

Key words

Acanthamoeba Dictyostelium Flow cytometry Green fluorescent protein Intracellular multiplication Legionella infection Macrophage Phagocytosis Type IV secretion 

Abbreviations

ACES

N-(2-Acetamido)-2-aminoethanesulfonic acid

icm/dot

Intracellular multiplication/defective organelle trafficking

MES

2-N-Morpholinoethanesulfonic

PI

Propidium iodide

T4SS

Type IV secretion system

FSC

Forward scatter channel

SSC

Sideward scatter channel

GFP

Green fluorescent protein

References

  1. 1.
    Rowbotham TJ (1980) Preliminary report on the pathogenicity of Legionella pneumophila for freshwater and soil amoebae. J Clin Pathol 33:1179–1183PubMedCrossRefGoogle Scholar
  2. 2.
    Hilbi H, Hoffmann C, Harrison CF (2011) Legionella spp. outdoors: colonization, communication and persistence. Environ Microbiol Rep 3:286–296CrossRefGoogle Scholar
  3. 3.
    Molmeret M, Horn M, Wagner M, Santic M, Abu Kwaik Y (2005) Amoebae as training grounds for intracellular bacterial pathogens. Appl Environ Microbiol 71:20–28PubMedCrossRefGoogle Scholar
  4. 4.
    Hilbi H, Weber SS, Ragaz C, Nyfeler Y, Urwyler S (2007) Environmental predators as models for bacterial pathogenesis. Environ Microbiol 9:563–575PubMedCrossRefGoogle Scholar
  5. 5.
    Nash TW, Libby DM, Horwitz MA (1984) Interaction between the Legionnaires’ disease bacterium (Legionella pneumophila) and human alveolar macrophages. Influence of antibody, lymphokines, and hydrocortisone. J Clin Invest 74:771–782PubMedCrossRefGoogle Scholar
  6. 6.
    Newton HJ, Ang DK, van Driel IR, Hartland EL (2010) Molecular pathogenesis of infections caused by Legionella pneumophila. Clin Microbiol Rev 23:274–298PubMedCrossRefGoogle Scholar
  7. 7.
    Isberg RR, O’Connor TJ, Heidtman M (2009) The Legionella pneumophila replication vacuole: making a cosy niche inside host cells. Nat Rev Microbiol 7:13–24PubMedCrossRefGoogle Scholar
  8. 8.
    Molofsky AB, Swanson MS (2004) Differentiate to thrive: lessons from the Legionella pneumophila life cycle. Mol Microbiol 53:29–40PubMedCrossRefGoogle Scholar
  9. 9.
    Brüggemann H, Hagman A, Jules M, Sismeiro O, Dillies MA, Gouyette C, Kunst F, Steinert M, Heuner K, Coppee JY, Buchrieser C (2006) Virulence strategies for infecting phagocytes deduced from the in vivo transcriptional program of Legionella pneumophila. Cell Microbiol 8:1228–1240PubMedCrossRefGoogle Scholar
  10. 10.
    Spirig T, Tiaden A, Kiefer P, Buchrieser C, Vorholt JA, Hilbi H (2008) The Legionella autoinducer synthase LqsA produces an α-hydroxyketone signaling molecule. J Biol Chem 283:18113–18123PubMedCrossRefGoogle Scholar
  11. 11.
    Tiaden A, Spirig T, Weber SS, Brüggemann H, Bosshard R, Buchrieser C, Hilbi H (2007) The Legionella pneumophila response regulator LqsR promotes host cell interactions as an ­element of the virulence regulatory network controlled by RpoS and LetA. Cell Microbiol 9:2903–2920PubMedCrossRefGoogle Scholar
  12. 12.
    Tiaden A, Spirig T, Hilbi H (2010) Bacterial gene regulation by α-hydroxyketone signaling. Trends Microbiol 18:288–297PubMedCrossRefGoogle Scholar
  13. 13.
    Horwitz MA, Silverstein SC (1983) Intracellular multiplication of Legionnaires’ disease bacteria (Legionella pneumophila) in human monocytes is reversibly inhibited by erythromycin and rifampin. J Clin Invest 71:15–26PubMedCrossRefGoogle Scholar
  14. 14.
    Feeley JC, Gibson RJ, Gorman GW, Langford NC, Rasheed JK, Mackel DC, Baine WB (1979) Charcoal-yeast extract agar: primary isolation medium for Legionella pneumophila. J Clin Microbiol 10:437–441PubMedGoogle Scholar
  15. 15.
    Cocucci SM, Sussman M (1970) RNA in cytoplasmic and nuclear fractions of cellular slime mold amebas. J Cell Biol 45:399–407PubMedCrossRefGoogle Scholar
  16. 16.
    Solomon JM, Isberg RR (2000) Growth of Legionella pneumophila in Dictyostelium discoideum: a novel system for genetic analysis of host-pathogen interactions. Trends Microbiol 8:478–480PubMedCrossRefGoogle Scholar
  17. 17.
    Malchow D, Nagele B, Schwarz H, Gerisch G (1972) Membrane-bound cyclic AMP phosphodiesterase in chemotactically responding cells of Dictyostelium discoideum. Eur J Biochem 28:136–142PubMedCrossRefGoogle Scholar
  18. 18.
    Moffat JF, Tompkins LS (1992) A quantitative model of intracellular growth of Legionella pneumophila in Acanthamoeba castellanii. Infect Immun 60:296–301PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  • André N. Tiaden
    • 1
  • Aline Kessler
    • 2
  • Hubert Hilbi
    • 2
  1. 1.Center for Applied Biotechnology and Molecular MedicineUniversity of ZurichZurichSwitzerland
  2. 2.Max von Pettenkofer InstituteLudwig-Maximilians UniversityMunichGermany

Personalised recommendations