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Analysis of Kinesin Accumulation on Salmonella-Containing Vacuoles

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Salmonella

Part of the book series: Methods in Molecular Biology ((MIMB,volume 394))

Abstract

Salmonella enterica is an intracellular bacterial pathogen that causes gastroenteritis and typhoid fever. Inside host cells, the bacterium is enclosed in a membrane bound compartment, the Salmonella-containing vacuole (SCV). Intracellular replication of Salmonella requires the translocation of effector proteins into the host cytosol. The SifA effector protein is important for the membrane stability of the SCV. Recently, we have shown that the Salmonella sifA mutant presents on its vacuole an important accumulation of kinesin-1, a molecular motor involved in the plus-end-directed transport of various organelles. Kinesin-1 is not recruited on SCVs of mutants that do not translocate effector proteins. This indicates that SifA is a negative regulator of the recruitment of this molecular motor and reveals the existence of another effector that recruits kinesin-1. This chapter describes techniques that are used to screen by immunofluorescence microscopy the accumulation of kinesin-1 on strains of Salmonella carrying multiple mutations.

These authors contributed equally to this work.

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References

  1. Galan, J. E. and Zhou, D. (2000) Striking a balance: modulation of the actin cytoskeleton by Salmonella. Proc. Natl. Acad. Sci. USA 97, 8754–87561.

    Article  CAS  PubMed  Google Scholar 

  2. Garcia-del Portillo, F. and Finlay, B. B. (1995) The varied lifestyles of intracellular pathogens within eukaryotic vacuolar compartments. Trends Microbiol. 3, 373–380.

    Article  CAS  PubMed  Google Scholar 

  3. Hensel, M., Shea, J. E., Gleeson, C., Jones, M. D., Dalton, E., and Holden, D. W. (1995) Simultaneous identification of bacterial virulence genes by negative selection. Science 269, 400–403.

    Article  CAS  PubMed  Google Scholar 

  4. Steele-Mortimer, O., Méresse, S., Gorvel, J. P., Toh, B. H., and Finlay, B. B. (1999) Biogenesis of Salmonella typhimurium-containing vacuoles in epithelial cells involves interactions with the early endocytic pathway. Cell Microbiol. 1, 33–49.

    Article  CAS  PubMed  Google Scholar 

  5. Méresse, S., Steele-Mortimer, O., Finlay, B. B., and Gorvel, J.-P. (1999) The rab7 GTPase controls the maturation of Salmonella typhimurium-containing vacuole in HeLa cells. EMBO J. 18, 4394–4403.

    Article  PubMed  Google Scholar 

  6. Beuzon, C. R., Méresse, S., Unsworth, K. E., et al. (2000) Salmonella maintains the integrity of its intracellular vacuole through the action of SifA. EMBO J. 19, 3235–3249.

    Article  CAS  PubMed  Google Scholar 

  7. Garcia-del Portillo, F. and Finlay, B. B. (1995) Targeting of Salmonella typhimurium to vesicles containing lysosomal membrane glycoproteins bypasses compartments with mannose 6-phosphate receptors. J. Cell Biol. 129, 81–97.

    Article  CAS  PubMed  Google Scholar 

  8. Garcia-del Portillo, F., Zwick, M. B., Leung, K. Y., and Finlay, B. B. (1993) Salmonella induces the formation of filamentous structures containing lysosomal membrane glycoproteins in epithelial cells. Proc. Natl. Acad. Sci. USA 90, 10544–10548.

    Article  CAS  PubMed  Google Scholar 

  9. Brumell, J. H., Goosney, D. L., and Finlay, B. B. (2002) SifA, a type III secreted effector of Salmonella typhimurium, directs Salmonella-induced filament (Sif) formation along microtubules. Traffic 3, 407–415.

    Article  CAS  PubMed  Google Scholar 

  10. Brumell, J. H., Rosenberger, C. M., Gotto, G. T., Marcus, S. L., and Finlay, B. B. (2001) SifA permits survival and replication of Salmonella typhimurium in murine macrophages. Cell Microbiol. 3, 75–84.

    Article  CAS  PubMed  Google Scholar 

  11. Stein, M. A., Leung, K. Y., Zwick, M., Garcia-del Portillo, F., and Finlay, B. B. (1996) Identification of a Salmonella virulence gene required for formation of filamentous structures containing lysosomal membrane glycoproteins within epithelial cells. Mol. Microbiol. 20, 151–164.

    Article  CAS  PubMed  Google Scholar 

  12. Tanaka, Y., Kanai, Y., Okada, Y., et al. (1998) Targeted disruption of mouse conventional kinesin heavy chain, kif5B, results in abnormal perinuclear clustering of mitochondria. Cell 93, 1147–1158.

    Article  CAS  PubMed  Google Scholar 

  13. Lippincott-Schwartz, J., Cole, N. B., Marotta, A., Conrad, P. A., and Bloom, G. S. (1995) Kinesin is the motor for microtubule-mediated Golgi-to-ER membrane traffic. J. Cell Biol. 128, 293–306.

    Article  CAS  PubMed  Google Scholar 

  14. Guignot, J., Caron, E., Beuzon, C., et al. (2004) Microtubule motors control membrane dynamics of Salmonella-containing vacuoles. J. Cell Sci. 117, 1033–1045.

    Article  CAS  PubMed  Google Scholar 

  15. Boucrot, E., Henry, T., Borg, J. P., Gorvel, J. P., and Meresse, S. (2005) The intracellular fate of Salmonella depends on the recruitment of kinesin. Science 308, 1174–1178.

    Article  CAS  PubMed  Google Scholar 

  16. Datsenko, K. A. and Wanner, B. L. (2000) One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl. Acad. Sci. USA 97, 6640–6645.

    Article  CAS  PubMed  Google Scholar 

  17. Schmieger, H. and Buch, U. (1975) Appearance of transducing particles and the fate of host DNA after infection of Salmonella typhimurium with P22-mutants with increased transducing ability (HT-mutants). Mol. Gen. Genet. 140, 111–122.

    Article  CAS  PubMed  Google Scholar 

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© 2007 Humana Press Inc., Totowa, NJ

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Dumont, A., Schroeder, N., Gorvel, JP., Méresse, S. (2007). Analysis of Kinesin Accumulation on Salmonella-Containing Vacuoles. In: Schatten, H., Eisenstark, A. (eds) Salmonella. Methods in Molecular Biology, vol 394. Humana Press. https://doi.org/10.1007/978-1-59745-512-1_13

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  • DOI: https://doi.org/10.1007/978-1-59745-512-1_13

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-58829-619-1

  • Online ISBN: 978-1-59745-512-1

  • eBook Packages: Springer Protocols

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