1887

Abstract

Members of the genus are facultative intracellular pathogenic bacteria able to control maturation of their vacuoles. In several cell types, is able to reach a proliferation compartment derived from the endoplasmic reticulum (ER). Since ER exit site (ERES) functions are required for proliferation, we performed a yeast two-hybrid screen between human ERES-associated proteins and the predicted brucella proteome. This screening led to the identification of CstA, a conserved protein that specifically interacts with Sec24A, a component of the ERES. We found that a tagged CstA is secreted in culture medium. This secretion is independent of the type IV secretion system VirB and the flagellum, suggesting that CstA is secreted through another system. We also discovered that a mutant is impaired for its association with the Sec23 ERES marker. The mutant displayed peculiar trafficking, with reduced association with LAMP1 and Calnexin 12 h post-infection in HeLa cells. However, its intracellular proliferation kinetics was not affected. The data reported here suggest that CstA could be directly or indirectly involved in the control of intracellular trafficking in HeLa cells.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.060509-0
2012-10-01
2024-03-29
Loading full text...

Full text loading...

/deliver/fulltext/micro/158/10/2610.html?itemId=/content/journal/micro/10.1099/mic.0.060509-0&mimeType=html&fmt=ahah

References

  1. Arenas G. N., Staskevich A. S., Aballay A., Mayorga L. S. ( 2000). Intracellular trafficking of Brucella abortus in J774 macrophages. Infect Immun 68:4255–4263 [View Article][PubMed]
    [Google Scholar]
  2. Bellaire B. H., Roop R. M. II, Cardelli J. A. ( 2005). Opsonized virulent Brucella abortus replicates within nonacidic, endoplasmic reticulum-negative, LAMP-1-positive phagosomes in human monocytes. Infect Immun 73:3702–3713 [View Article][PubMed]
    [Google Scholar]
  3. Celli J., de Chastellier C., Franchini D. M., Pizarro-Cerda J., Moreno E., Gorvel J. P. ( 2003). Brucella evades macrophage killing via VirB-dependent sustained interactions with the endoplasmic reticulum. J Exp Med 198:545–556 [View Article][PubMed]
    [Google Scholar]
  4. Celli J., Salcedo S. P., Gorvel J. P. ( 2005). Brucella coopts the small GTPase Sar1 for intracellular replication. Proc Natl Acad Sci U S A 102:1673–1678 [View Article][PubMed]
    [Google Scholar]
  5. Cloeckaert A., Jacques I., Bowden R. A., Dubray G., Limet J. N. ( 1993a). Monoclonal antibodies to Brucella rough lipopolysaccharide: characterization and evaluation of their protective effect against B. abortus . Res Microbiol 144:475–484 [View Article][PubMed]
    [Google Scholar]
  6. Cloeckaert A., Zygmunt M. S., Dubray G., Limet J. N. ( 1993b). Characterization of O-polysaccharide specific monoclonal antibodies derived from mice infected with the rough Brucella melitensis strain B115. J Gen Microbiol 139:1551–1556 [View Article][PubMed]
    [Google Scholar]
  7. de Barsy M., Jamet A., Filopon D., Nicolas C., Laloux G., Rual J. F., Muller A., Twizere J. C., Nkengfac B. & other authors ( 2011). Identification of a Brucella spp. secreted effector specifically interacting with human small GTPase Rab2. Cell Microbiol 13:1044–1058 [View Article][PubMed]
    [Google Scholar]
  8. de Jong M. F., Sun Y. H., den Hartigh A. B., van Dijl J. M., Tsolis R. M. ( 2008). Identification of VceA and VceC, two members of the VjbR regulon that are translocated into macrophages by the Brucella type IV secretion system. Mol Microbiol 70:1378–1396 [View Article][PubMed]
    [Google Scholar]
  9. Dricot A., Rual J. F., Lamesch P., Bertin N., Dupuy D., Hao T., Lambert C., Hallez R., Delroisse J. M. & other authors ( 2004). Generation of the Brucella melitensis ORFeome version 1.1. Genome Res 14:10B2201–2206 [View Article][PubMed]
    [Google Scholar]
  10. Fugier E., Salcedo S. P., de Chastellier C., Pophillat M., Muller A., Arce-Gorvel V., Fourquet P., Gorvel J. P. ( 2009). The glyceraldehyde-3-phosphate dehydrogenase and the small GTPase Rab2 are crucial for Brucella replication. PLoS Pathog 5:e1000487 [View Article][PubMed]
    [Google Scholar]
  11. Garin-Bastuji B., Bowden R. A., Dubray G., Limet J. N. ( 1990). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting analysis of smooth-lipopolysaccharide heterogeneity among Brucella biovars related to A and M specificities. J Clin Microbiol 28:2169–2174[PubMed]
    [Google Scholar]
  12. Godfroid F., Taminiau B., Danese I., Denoel P., Tibor A., Weynants V., Cloeckaert A., Godfroid J., Letesson J. J. ( 1998). Identification of the perosamine synthetase gene of Brucella melitensis 16M and involvement of lipopolysaccharide O side chain in Brucella survival in mice and in macrophages. Infect Immun 66:5485–5493[PubMed]
    [Google Scholar]
  13. González D., Grilló M. J., De Miguel M. J., Ali T., Arce-Gorvel V., Delrue R. M., Conde-Alvarez R., Muñoz P., López-Goñi I. & other authors ( 2008). Brucellosis vaccines: assessment of Brucella melitensis lipopolysaccharide rough mutants defective in core and O-polysaccharide synthesis and export. PLoS ONE 3:e2760 [View Article][PubMed]
    [Google Scholar]
  14. Haag A. F., Myka K. K., Arnold M. F., Caro-Hernández P., Ferguson G. P. ( 2010). Importance of lipopolysaccharide and cyclic β-1,2-glucans in Brucella–mammalian infections. Int J Microbiol 2010:124509[PubMed] [CrossRef]
    [Google Scholar]
  15. Hackstadt T., Williams J. C. ( 1981). Biochemical stratagem for obligate parasitism of eukaryotic cells by Coxiella burnetii . Proc Natl Acad Sci U S A 78:3240–3244 [View Article][PubMed]
    [Google Scholar]
  16. Heinzen R. A., Scidmore M. A., Rockey D. D., Hackstadt T. ( 1996). Differential interaction with endocytic and exocytic pathways distinguish parasitophorous vacuoles of Coxiella burnetii and Chlamydia trachomatis . Infect Immun 64:796–809[PubMed]
    [Google Scholar]
  17. Isberg R. R., O’Connor T. J., Heidtman M. ( 2009). The Legionella pneumophila replication vacuole: making a cosy niche inside host cells. Nat Rev Microbiol 7:13–24 [View Article][PubMed]
    [Google Scholar]
  18. James P., Halladay J., Craig E. A. ( 1996). Genomic libraries and a host strain designed for highly efficient two-hybrid selection in yeast. Genetics 144:1425–1436[PubMed]
    [Google Scholar]
  19. Lamesch P., Li N., Milstein S., Fan C., Hao T., Szabo G., Hu Z., Venkatesan K., Bethel G. & other authors ( 2007). hORFeome v3.1: a resource of human open reading frames representing over 10,000 human genes. Genomics 89:307–315 [View Article][PubMed]
    [Google Scholar]
  20. Mancias J. D., Goldberg J. ( 2005). Exiting the endoplasmic reticulum. Traffic 6:278–285 [View Article][PubMed]
    [Google Scholar]
  21. Marchesini M. I., Herrmann C. K., Salcedo S. P., Gorvel J. P., Comerci D. J. ( 2011). In search of Brucella abortus type IV secretion substrates: screening and identification of four proteins translocated into host cells through VirB system. Cell Microbiol 13:1261–1274 [View Article][PubMed]
    [Google Scholar]
  22. Mignolet J., Van der Henst C., Nicolas C., Deghelt M., Dotreppe D., Letesson J. J., De Bolle X. ( 2010). PdhS, an old-pole-localized histidine kinase, recruits the fumarase FumC in Brucella abortus . J Bacteriol 192:3235–3239 [View Article][PubMed]
    [Google Scholar]
  23. Monreal D., Grilló M. J., González D., Marín C. M., De Miguel M. J., López-Goñi I., Blasco J. M., Cloeckaert A., Moriyón I. ( 2003). Characterization of Brucella abortus O-polysaccharide and core lipopolysaccharide mutants and demonstration that a complete core is required for rough vaccines to be efficient against Brucella abortus and Brucella ovis in the mouse model. Infect Immun 71:3261–3271 [View Article][PubMed]
    [Google Scholar]
  24. Ninio S., Roy C. R. ( 2007). Effector proteins translocated by Legionella pneumophila: strength in numbers. Trends Microbiol 15:372–380 [View Article][PubMed]
    [Google Scholar]
  25. Pizarro-Cerdá J., Moreno E., Sanguedolce V., Mege J.-L., Gorvel J.-P. ( 1998). Virulent Brucella abortus prevents lysosome fusion and is distributed within autophagosome-like compartments. Infect Immun 66:2387–2392[PubMed]
    [Google Scholar]
  26. Quandt J., Hynes M. F. ( 1993). Versatile suicide vectors which allow direct selection for gene replacement in Gram negative bacteria. Gene 127:15–21[PubMed] [CrossRef]
    [Google Scholar]
  27. Rual J. F., Hirozane-Kishikawa T., Hao T., Bertin N., Li S., Dricot A., Li N., Rosenberg J., Lamesch P. & other authors ( 2004). Human ORFeome version 1.1: a platform for reverse proteomics. Genome Res 14:10B2128–2135 [View Article][PubMed]
    [Google Scholar]
  28. Sambrook J., Russell D. W. ( 2001). Molecular cloning, a laboratory manual, 3rd edn. Cold Spring Harbour, NY: Cold Spring Harbour Laboratory;
    [Google Scholar]
  29. Sherman F. ( 1991). Getting started with yeast. Methods Enzymol 194:3–21 [View Article][PubMed]
    [Google Scholar]
  30. Simon R., Priefer U., Pühler A. ( 1983). A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in Gram negative bacteria. Nat Biotechnol 1:784–791 [View Article]
    [Google Scholar]
  31. Vergne I., Chua J., Singh S. B., Deretic V. ( 2004). Cell biology of Mycobacterium tuberculosis phagosome. Annu Rev Cell Dev Biol 20:367–394 [View Article][PubMed]
    [Google Scholar]
  32. Vidalain P. O., Boxem M., Ge H., Li S., Vidal M. ( 2004). Increasing specificity in high-throughput yeast two-hybrid experiments. Methods 32:363–370 [View Article][PubMed]
    [Google Scholar]
  33. Walhout A. J., Vidal M. ( 2001). High-throughput yeast two-hybrid assays for large-scale protein interaction mapping. Methods 24:297–306 [View Article][PubMed]
    [Google Scholar]
  34. Wendeler M. W., Paccaud J. P., Hauri H. P. ( 2007). Role of Sec24 isoforms in selective export of membrane proteins from the endoplasmic reticulum. EMBO Rep 8:258–264 [View Article][PubMed]
    [Google Scholar]
  35. Young G. M., Schmiel D. H., Miller V. L. ( 1999). A new pathway for the secretion of virulence factors by bacteria: the flagellar export apparatus functions as a protein-secretion system. Proc Natl Acad Sci U S A 96:6456–6461 [View Article][PubMed]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.060509-0
Loading
/content/journal/micro/10.1099/mic.0.060509-0
Loading

Data & Media loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error