Skip to main content
SpringerLink
Log in
Book cover

Legionella pp 323–332Cite as

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

  • Protocol
  • First Online:

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

Abstract

Legionella pneumophila the causative agent of Legionnaires’ disease, actively manipulates host cell ­processes to establish a membrane-bound replication vacuole permissive for its replication. Establishment of such replication niche requires the Dot/Icm type IV secretion system which translocates a plethora of effectors into host cells. Determining whether a particular protein is a substrate of the transporter is a prerequisite for subsequent functional studies. Thus, a variety of methods have been developed in the last decade to measure Dot/Icm-dependent delivery of protein into the host cell. The combination of these methods and the appropriate screening strategies has allowed for the identification of more than 270 translocated proteins. These efforts have laid a solid foundation for further study of the roles of these proteins in the interactions between L. pneumophila and its host. Here, we summarized the experimental details of these methods.

This is a preview of subscription content, 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

Learn about institutional subscriptions

Springer Nature is developing a new tool to find and evaluate Protocols. Learn more

References

  1. Fields BS, Benson RF, Besser RE (2002) Legionella and Legionnaires’ disease: 25 years of investigation. Clin Microbiol Rev 15:506–526

    Article  PubMed  Google Scholar 

  2. Horwitz MA (1983) The Legionnaires’ disease bacterium (Legionella pneumophila) inhibits phagosome-lysosome fusion in human monocytes. J Exp Med 158:2108–2126

    Article  PubMed  CAS  Google Scholar 

  3. Tilney LG, Harb OS, Connelly PS, Robinson CG, Roy CR (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

    PubMed  CAS  Google Scholar 

  4. Isberg RR, O’Connor TJ, Heidtman M (2009) The Legionella pneumophila replication vacuole: making a cosy niche inside host cells. Nat Rev Microbiol 7:13–24

    Article  PubMed  CAS  Google Scholar 

  5. Vogel JP, Andrews HL, Wong SK, Isberg RR (1998) Conjugative transfer by the virulence system of Legionella pneumophila. Science 279:873–876

    Article  PubMed  CAS  Google Scholar 

  6. Segal G, Purcell M, Shuman HA (1998) Host cell killing and bacterial conjugation require overlapping sets of genes within a 22–kb region of the Legionella pneumophila genome. Proc Natl Acad Sci USA 95:1669–1674

    Article  PubMed  CAS  Google Scholar 

  7. Luo ZQ Isberg RR (2004) Multiple substrates of the Legionella pneumophila Dot/Icm system identified by interbacterial protein transfer. Proc Natl Acad Sci USA101:841–846

    Article  PubMed  CAS  Google Scholar 

  8. Nagai H, Kagan JC, Zhu X, Kahn RA, Roy CR (2002) A bacterial guanine nucleotide exchange factor activates ARF on Legionella phagosomes. Science 295:679–682

    Article  PubMed  CAS  Google Scholar 

  9. Huang L, et al. (2010) The E Block motif is associated with Legionella pneumophila translocated substrates. Cell Microbiol 13:227–245

    Article  PubMed  Google Scholar 

  10. VanRheenen SM, Luo ZQ, O’Connor T, Isberg RR (2006) Members of a Legionella pneumophila family of proteins with ExoU (phospholipase A) active sites are translocated to target cells. Infect Immun 74:3597–3606

    Article  PubMed  CAS  Google Scholar 

  11. Chen J, et al. (2004) Legionella effectors that promote nonlytic release from protozoa. Science 303:1358–1361

    Article  PubMed  CAS  Google Scholar 

  12. Sory MP, Cornelis GR (1994) Translocation of a hybrid YopE–adenylate cyclase from Yersinia enterocolitica into HeLa cells. Mol Microbiol 14:583–594

    Article  PubMed  CAS  Google Scholar 

  13. Glaser P, Sakamoto H, Bellalou J, Ullmann A, Danchin A (1988) Secretion of cyclolysin, the calmodulin–sensitive adenylate cyclase–haemolysin bifunctional protein of Bordetella pertussis. EMBO J 7:3997–4004

    PubMed  CAS  Google Scholar 

  14. de Felipe KS, et al. (2005) Evidence for acquisition of Legionella type IV secretion substrates via interdomain horizontal gene transfer. J Bacteriol 187:7716–7726

    Article  PubMed  Google Scholar 

  15. Zusman T, Degtyar E, Segal G (2008) Identification of a hypervariable region containing new Legionella pneumophila Icm/Dot translocated substrates by using the conserved icmQ regulatory signature. Infect Immun 76:4581–4591

    Article  PubMed  CAS  Google Scholar 

  16. Burstein D, et al. (2009) Genome-scale identification of Legionella pneumophila effectors using a machine learning approach. PLoS Pathog 5:e1000508

    Article  PubMed  Google Scholar 

  17. Kubori T, Hyakutake A, Nagai H (2008) Legionella translocates an E3 ubiquitin ligase that has multiple U-boxes with distinct functions. Mol Microbiol 67:1307–1319

    Article  PubMed  CAS  Google Scholar 

  18. Ninio S, Celli J, Roy CR (2009) A Legionella pneumophila effector protein encoded in a region of genomic plasticity binds to Dot/Icm-modified vacuoles. PLoS Pathog5:e1000278

    Article  PubMed  Google Scholar 

  19. Altman E Segal G (2008) The response regulator CpxR directly regulates expression of several Legionella pneumophila icm/dot components as well as new translocated substrates. J Bacteriol 190:1985–1996

    Article  PubMed  CAS  Google Scholar 

  20. Pan X, Luhrmann A, Satoh A, Laskowski-Arce MA, Roy CR (2008) Ankyrin repeat proteins comprise a diverse family of bacterial type IV effectors. Science 320:1651–1654

    Article  PubMed  CAS  Google Scholar 

  21. Zusman T, et al. (2007) The response regulator PmrA is a major regulator of the icm/dot type IV secretion system in Legionella pneumophila and Coxiella burnetii. Mol Microbiol63:1508–1523

    Article  PubMed  CAS  Google Scholar 

  22. Charpentier X Oswald E (2004) Identification of the secretion and translocation domain of the enteropathogenic and enterohemorrhagic Escherichia coli effector Cif, using TEM-1 beta-lactamase as a new fluorescence-based reporter. J Bacteriol 186:5486–5495

    Article  PubMed  CAS  Google Scholar 

  23. de Felipe KS, et al. (2008) Legionella eukaryotic-like type IV substrates interfere with organelle trafficking. PLoS Pathog4:e1000117

    Article  PubMed  Google Scholar 

  24. Charpentier X, et al. (2009) Chemical genetics reveals bacterial and host cell functions critical for type IV effector translocation by Legionella pneumophila. PLoS Pathog 5:e1000501

    Article  PubMed  Google Scholar 

  25. Zhu W, et al. (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 

  26. 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 Pathog2:e46

    Article  PubMed  Google Scholar 

  27. Bardill JP, Miller JL, Vogel JP (2005) IcmS-dependent translocation of SdeA into macrophages by the Legionella pneumophila type IV secretion system. Mol Microbiol56:90–103

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Biological Sciences, Purdue University, West Lafayette, IN, USA

    Wenhan Zhu & Zhao-Qing Luo

Authors
  1. Wenhan Zhu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Zhao-Qing Luo
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Zhao-Qing Luo .

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

Zhu, W., Luo, ZQ. (2013). Methods for Determining Protein Translocation by the Legionella pneumophila Dot/Icm Type IV Secretion System. 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_19

Download citation

  • DOI: https://doi.org/10.1007/978-1-62703-161-5_19

  • 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