Skip to main content

Analyzing Association of the Endoplasmic Reticulum with the Legionella pneumophila–Containing Vacuoles by Fluorescence Microscopy

  • Protocol
Autophagosome and Phagosome

Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 445))

Summary

A unique feature of the intracellular life cycle of Legionella pneumophila is the interaction between the vacuole in which L. pneumophila resides and the endoplasmic reticulum of the host cell. This interaction is crucial for L. pneumophila to establish a niche in which the bacteria can replicate intracellularly. Microscopic analysis of endoplasmic reticulum (ER) markers during infection yields information regarding the nature of the recruited vesicles as well as the kinetics of their recruitment. The recruitment of YFP-KDEL, GFP-p58, calnexin, and myc-Sec22b to the L. pneumophila – containing vacuole can be assessed by fluorescence microscopy. Methods for detection of these various ER markers during infection of mammalian cells by L. pneumophila are described.

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

Access this chapter

Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 109.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 109.99
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

References

  1. Rowbotham, T. J. (1980) Preliminary report on the pathogenicity of Legionella pneumophila for freshwater and soil amoebae. J. Clin. Pathol. 33, 1179–1183.

    Article  CAS  PubMed  Google Scholar 

  2. Fraser, D. W., Tsai, T. R., Orenstein, W., et al. (1977) Legionnaires’ disease: description of an epidemic of pneumonia. N. Engl. J. Med. 297, 1189–1197.

    Article  CAS  PubMed  Google Scholar 

  3. McDade, J. E., Shepard, C. C., Fraser, D. W., Tsai, T. R., Redus, M. A., and Dowdle, W. R. (1977) Legionnaires’ disease: isolation of a bacterium and demonstration of its role in other respiratory disease. N. Engl. J. Med. 297, 1197–1203.

    Article  CAS  PubMed  Google Scholar 

  4. Horwitz, M. A., and Silverstein, S. C. (1980) Legionnaires’ disease bacterium (Legionella pneumophila) multiples intracellularly in human monocytes. J. Clin. Invest. 66, 441–450.

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  6. Horwitz, M. A. (1983) The Legionnaires’ disease bacterium (Legionella pneumophila) inhibits phagosome-lysosome fusion in human monocytes. J. Exp. Med. 158, 2108–2126.

    Article  CAS  PubMed  Google Scholar 

  7. Roy, C. R., Berger, K. H., and Isberg, R. R. (1998) Legionella pneumophila DotA protein is required for early phagosome trafficking decisions that occur within minutes of bacterial uptake. Mol. Microbiol. 28, 663–674.

    Article  CAS  PubMed  Google Scholar 

  8. Wiater, L. A., Dunn, K., Maxfield, F. R., and Shuman, H. A. (1998) Early events in phagosome establishment are required for intracellular survival of Legionella pneumophila. Infect. Immun. 66, 4450–4460.

    CAS  PubMed  Google Scholar 

  9. Swanson, M. S., and Isberg, R. R. (1995) Association of Legionella pneumophila with the macrophage endoplasmic reticulum. Infect. Immun. 63, 3609–3620.

    CAS  PubMed  Google Scholar 

  10. Tilney, L. G., Harb, O. S., Connelly, P. S., Robinson, C. G., and Roy, C. R. (2001) How the parasitic bacterium Legionella pneumophila modifies its phagosome and transforms it into rough ER: implications for conversion of plasma membrane to the ER membrane. J. Cell Sci. 114, 4637–4650.

    CAS  PubMed  Google Scholar 

  11. Robinson, C. G., and Roy, C. R. (2006) Attachment and fusion of endoplasmic reticulum with vacuoles containing Legionella pneumophila. Cell. Microbiol. 8, 793–805.

    Article  CAS  PubMed  Google Scholar 

  12. Kagan, J. C., and Roy, C. R. (2002) Legionella phagosomes intercept vesicular traffic from endoplasmic reticulum exit sites. Nat. Cell Biol. 4, 945–9454.

    Article  CAS  PubMed  Google Scholar 

  13. Kagan, J. C., Stein, M. P., Pypaert, M., and Roy, C. R. (2004) Legionella subvert the functions of rab1 and sec22b to create a replicative organelle. J. Exp. Med. 199, 1201–1211.

    Article  CAS  PubMed  Google Scholar 

  14. Derre, I., and Isberg, R. R. (2004) Legionella pneumophila replication vacuole formation involves rapid recruitment of proteins of the early secretory system. Infect. Immun. 72, 3048–3053.

    Article  CAS  PubMed  Google Scholar 

  15. Joiner, K. A., Fuhrman, S. A., Miettinen, H. M., Kasper, L. H., and Mellman, I. (1990) Toxoplasma gondii: fusion competence of parasitophorous vacuoles in Fc receptor-transfected fibroblasts. Science 249, 641–646.

    Article  CAS  PubMed  Google Scholar 

  16. Evan, G. I., Lewis, G. K., Ramsay, G., and Bishop, J. M. (1985) Isolation of monoclonal antibodies specific for human c-myc proto-oncogene product. Mol. Cell Biol. 5, 3610–3616.

    CAS  PubMed  Google Scholar 

  17. Celada, A., Gray, P. W., Rinderknecht, E., and Schreiber, R. D. (1984) Evidence for a gamma-interferon receptor that regulates macrophage tumoricidal activity. J. Exp. Med. 160, 55–74.

    Article  CAS  PubMed  Google Scholar 

  18. Naviaux, R. K., Costanzi, E., Haas, M., and Verma, I. M. (1996) The pCL vector system: rapid production of helper-free, high-titer, recombinant retroviruses. J. Virol. 70, 5701–5705.

    CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2008 Humana Press, a part of Springer Science+Business Media, LLC

About this protocol

Cite this protocol

Ingmundson, A., Roy, C.R. (2008). Analyzing Association of the Endoplasmic Reticulum with the Legionella pneumophila–Containing Vacuoles by Fluorescence Microscopy. In: Deretic, V. (eds) Autophagosome and Phagosome. Methods in Molecular Biology™, vol 445. Humana Press. https://doi.org/10.1007/978-1-59745-157-4_24

Download citation

  • DOI: https://doi.org/10.1007/978-1-59745-157-4_24

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-58829-853-9

  • Online ISBN: 978-1-59745-157-4

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics