Skip to main content
SpringerLink
Log in

Pathogen Vacuole Purification from Legionella-Infected Amoeba and Macrophages

  • Protocol
  • First Online:
Legionella

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

Abstract

Legionella pneumophila replicates intracellularly in environmental and immune phagocytes within a unique membrane-bound compartment, the Legionella-containing vacuole (LCV). Formation of LCVs is strictly dependent on the Icm/Dot type IV secretion system and the translocation of “effector” proteins into the cell. Some effector proteins decorate the LCV membrane and subvert host cell vesicle trafficking pathways. Here we describe a method to purify intact LCVs from Dictyostelium discoideum amoebae and RAW 264.7 murine macrophages. The method comprises a two-step protocol: first, LCVs are enriched by immuno-magnetic separation using an antibody against a bacterial effector protein specifically localizing to the LCV membrane, and second, the LCVs are further purified by density gradient centrifugation. The purified LCVs can be characterized by proteomics and other biochemical approaches.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 129.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 179.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

Abbreviations

ACES:

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

GFP:

Green fluorescent protein

HEPES:

N-2-Hydroxy-ethylpiperazine-N ¢-2-ethanesulfonic acid

Icm/Dot:

Intracellular multiplication/defective organelle trafficking

T4SS:

Type IV secretion system

References

  1. Newton HJ, Ang DK, van Driel IR, Hartland EL (2010) Molecular pathogenesis of infections caused by Legionella pneumophila. Clin Microbiol Rev 23:274–298

    Article  PubMed  CAS  Google Scholar 

  2. Hilbi H, Hoffmann C, Harrison CF (2011) Legionella spp. outdoors: colonization, communication and persistence. Environ Microbiol Rep 3:286–296

    Article  CAS  Google Scholar 

  3. Zhu W, Banga S, Tan Y, Zheng C, Stephenson R, Gately J, Luo ZQ (2011) Comprehensive identification of protein substrates of the Dot/Icm type IV transporter of Legionella pneumophila. PLoS One 6:e17638

    Article  PubMed  CAS  Google Scholar 

  4. Hubber A, Roy CR (2010) Modulation of host cell function by Legionella pneumophila type IV effectors. Annu Rev Cell Dev Biol 26:261–283

    Article  PubMed  CAS  Google Scholar 

  5. Garin J, Diez R, Kieffer S, Dermine JF, Duclos S, Gagnon E, Sadoul R, Rondeau C, Desjardins M (2001) The phagosome proteome: insight into phagosome functions. J Cell Biol 152:165–180

    Article  PubMed  CAS  Google Scholar 

  6. Gotthardt D, Dieckmann R, Blancheteau V, Kistler C, Reichardt F, Soldati T (2006) Preparation of intact, highly purified phagosomes from Dictyostelium. Methods Mol Biol 346:439–448

    PubMed  CAS  Google Scholar 

  7. Gotthardt D, Warnatz HJ, Henschel O, Bruckert F, Schleicher M, Soldati T (2002) High-resolution dissection of phagosome maturation reveals distinct membrane trafficking phases. Mol Biol Cell 13:3508–3520

    Article  PubMed  CAS  Google Scholar 

  8. Kima PE, Dunn W (2005) Exploiting calnexin expression on phagosomes to isolate Leishmania parasitophorous vacuoles. Microb Pathog 38:139–145

    Article  PubMed  CAS  Google Scholar 

  9. Lührmann A, Haas A (2000) A method to purify bacteria-containing phagosomes from infected macrophages. Methods Cell Sci 22:329–341

    Article  PubMed  Google Scholar 

  10. Sturgill-Koszycki S, Haddix PL, Russell DG (1997) The interaction between Mycobacterium and the macrophage analyzed by two-dimensional polyacrylamide gel electrophoresis. Electrophoresis 18:2558–2565

    Article  PubMed  CAS  Google Scholar 

  11. Fernandez-Mora E, Polidori M, Lührmann A, Schaible UE, Haas A (2005) Maturation of Rhodococcus equi-containing vacuoles is arrested after completion of the early endosome stage. Traffic 6:635–653

    Article  PubMed  CAS  Google Scholar 

  12. Mills SD, Finlay BB (1998) Isolation and characterization of Salmonella typhimurium and Yersinia pseudotuberculosis-containing phagosomes from infected mouse macrophages: Y. pseudotuberculosis traffics to terminal lysosomes where they are degraded. Eur J Cell Biol 77:35–47

    Article  PubMed  CAS  Google Scholar 

  13. Shevchuk O, Batzilla C, Hagele S, Kusch H, Engelmann S, Hecker M, Haas A, Heuner K, Glockner G, Steinert M (2009) Proteomic analysis of Legionella-containing phagosomes isolated from Dictyostelium. Int J Med Microbiol 299:489–508

    Article  PubMed  CAS  Google Scholar 

  14. Urwyler S, Nyfeler Y, Ragaz C, Lee H, Mueller LN, Aebersold R, Hilbi H (2009) Proteome analysis of Legionella vacuoles purified by magnetic immunoseparation reveals secretory and endosomal GTPases. Traffic 10:76–87

    Article  PubMed  CAS  Google Scholar 

  15. Urwyler S, Finsel I, Ragaz C, Hilbi H (2010) Isolation of Legionella-containing vacuoles by immuno-magnetic separation. Curr Protoc Cell Biol Chapter 3, Unit 3.34

    Google Scholar 

  16. Brombacher E, Urwyler S, Ragaz C, Weber SS, Kami K, Overduin M, Hilbi H (2009) Rab1 guanine nucleotide exchange factor SidM is a major phosphatidylinositol 4-phosphate-binding effector protein of Legionella pneumophila. J Biol Chem 284:4846–4856

    Article  PubMed  CAS  Google Scholar 

  17. Hilbi H, Weber S, Finsel I (2011) Anchors for effectors: subversion of phosphoinositide lipids by Legionella. Front Microbiol 2:91

    PubMed  CAS  Google Scholar 

  18. Urwyler S, Brombacher E, Hilbi H (2009) Endosomal and secretory markers of the Legionella-containing vacuole. Commun Integr Biol 2:107–109

    PubMed  CAS  Google Scholar 

  19. Mampel J, Spirig T, Weber SS, Haagensen JAJ, Molin S, Hilbi H (2006) Planktonic replication is essential for biofilm formation by Legionella pneumophila in a complex medium under static and dynamic flow conditions. Appl Environ Microbiol 72:2885–2895

    Article  PubMed  CAS  Google Scholar 

  20. Horwitz MA (1983) Formation of a novel phagosome by the Legionnaires’ disease bacterium (Legionella pneumophila) in human monocytes. J Exp Med 158:1319–1331

    Article  PubMed  CAS  Google Scholar 

  21. 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 10:437–441

    PubMed  CAS  Google Scholar 

  22. Müller-Taubenberger A, Lupas AN, Li H, Ecke M, Simmeth E, Gerisch G (2001) Calreticulin and calnexin in the endoplasmic reticulum are important for phagocytosis. EMBO J 20:6772–6782

    Article  PubMed  Google Scholar 

  23. Cocucci SM, Sussman M (1970) RNA in cytoplasmic and nuclear fractions of cellular slime mold amebas. J Cell Biol 45:399–407

    Article  PubMed  CAS  Google Scholar 

  24. Malchow D, Nagele B, Schwarz H, Gerisch G (1972) Membrane-bound cyclic AMP phosphodiesterase in chemotactically responding cells of Dictyostelium discoideum. Eur J Biochem 28:136–142

    Article  PubMed  CAS  Google Scholar 

  25. Weber SS, Ragaz C, Reus K, Nyfeler Y, Hilbi H (2006) Legionella pneumophila exploits PI(4)P to anchor secreted effector proteins to the replicative vacuole. PLoS Pathog 2:e46

    Article  PubMed  Google Scholar 

  26. Ragaz C, Pietsch H, Urwyler S, Tiaden A, Weber SS, Hilbi H (2008) The Legionella pneumophila phosphatidylinositol-4 phosphate-binding type IV substrate SidC recruits endoplasmic reticulum vesicles to a replication-permissive vacuole. Cell Microbiol 10:2416–2433

    Article  PubMed  CAS  Google Scholar 

  27. Lu H, Clarke M (2005) Dynamic properties of Legionella-containing phagosomes in Dictyostelium amoebae. Cell Microbiol 7:995–1007

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

This work was supported by the Max von Pettenkofer Institute, Ludwig-Maximilians University Munich, and the German Research Foundation (BMBF “Medical Infection Genomics”, HI 1511/3-1).

Author information

Authors and Affiliations

  1. Max von Pettenkofer Institute, Ludwig-Maximilians University, Munich, Germany

    Christine Hoffmann, Ivo Finsel & Hubert Hilbi

Authors
  1. Christine Hoffmann
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ivo Finsel
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Hubert Hilbi
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Hubert Hilbi .

Editor information

Editors and Affiliations

  1. , Biology of Intracellular Bacteria, Institut Pasteur, Rue du Dr. Roux 28, Paris, 75724, France

    Carmen Buchrieser

  2. Max von Pettenkofer Institute, Ludwig-Maximilians-University, Pettenkoferstrasse 9a, Munich, 80336, Germany

    Hubert Hilbi

Rights and permissions

Reprints and permissions

Copyright information

© 2013 Springer Science+Business Media New York

About this protocol

Cite this protocol

Hoffmann, C., Finsel, I., Hilbi, H. (2013). Pathogen Vacuole Purification from Legionella-Infected Amoeba and Macrophages. In: Buchrieser, C., Hilbi, H. (eds) Legionella. Methods in Molecular Biology, vol 954. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-161-5_18

Download citation

  • DOI: https://doi.org/10.1007/978-1-62703-161-5_18

  • Published:

  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-160-8

  • Online ISBN: 978-1-62703-161-5

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation