1887

Abstract

The type III secretion apparatus (T3SA) is a central virulence factor of many Gram-negative bacteria. Its overall morphology consists of a cytoplasmic region, inner- and outer-membrane sections and an extracellular needle. In , the length of the needle is regulated by Spa32. To understand better the role of Spa32 we searched for its interacting partners using a two-hybrid screen in yeast. We found that Spa32 interacts with the 33 C-terminal residues (CC*) of Spa40, a member of the conserved FlhB/YscU family. Using a GST pull-down assay we confirmed this interaction and identified additional interactions between Spa40 and the type III secretion components Spa33, Spa47, MxiK, MxiN and MxiA. Inactivation of abolished protein secretion and led to needleless structures. Genetic and functional analyses were used to investigate the roles of residues L310 and V320, located within the CC* domain of Spa40, in the assembly of the T3SA. Spa40 cleavage, at the conserved NPTH motif, is required for assembly of the T3SA and for its interaction with Spa32, Spa33 and Spa47. In contrast, unprocessed forms of Spa40 interacted only with MxiA, MxiK and MxiN. Our data suggest that the conformation of the cytoplasmic domain of Spa40 defines the multi-step assembly process of the T3SA.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.039651-0
2010-09-01
2020-07-11
Loading full text...

Full text loading...

/deliver/fulltext/micro/156/9/2807.html?itemId=/content/journal/micro/10.1099/mic.0.039651-0&mimeType=html&fmt=ahah

References

  1. Aizawa S. I.. 2001; Bacterial flagella and type III secretion systems. FEMS Microbiol Lett202:157–164
    [Google Scholar]
  2. Allaoui A., Mounier J., Prevost M. C., Sansonetti P. J., Parsot C.. 1992a; icsB: a Shigella flexneri virulence gene necessary for the lysis of protrusions during intercellular spread. Mol Microbiol6:1605–1616
    [Google Scholar]
  3. Allaoui A., Sansonetti P. J., Parsot C.. 1992b; MxiJ, a lipoprotein involved in secretion of Shigella Ipa invasins, is homologous to YscJ, a secretion factor of the Yersinia Yop proteins. J Bacteriol174:7661–7669
    [Google Scholar]
  4. Allaoui A., Menard R., Sansonetti P. J., Parsot C.. 1993a; Characterization of the Shigella flexneri ipgD and ipgF genes, which are located in the proximal part of the mxi locus. Infect Immun61:1707–1714
    [Google Scholar]
  5. Allaoui A., Sansonetti P. J., Parsot C.. 1993b; MxiD, an outer membrane protein necessary for the secretion of the Shigella flexneri Ipa invasins. Mol Microbiol7:59–68
    [Google Scholar]
  6. Allaoui A., Woestyn S., Sluiters C., Cornelis G. R.. 1994; YscU, a Yersinia enterocolitica inner membrane protein involved in Yop secretion. J Bacteriol176:4534–4542
    [Google Scholar]
  7. Allaoui A., Sansonetti P. J., Menard R., Barzu S., Mounier J., Phalipon A., Parsot C.. 1995; MxiG, a membrane protein required for secretion of Shigella spp. Ipa invasins: involvement in entry into epithelial cells and in intercellular dissemination. Mol Microbiol17:461–470
    [Google Scholar]
  8. Andrews G. P., Maurelli A. T.. 1992; mxiA of Shigella flexneri 2a, which facilitates export of invasion plasmid antigens, encodes a homolog of the low-calcium-response protein, LcrD, of Yersinia pestis. Infect Immun60:3287–3295
    [Google Scholar]
  9. Barzu S., Nato F., Rouyre S., Mazie J. C., Sansonetti P. J., Phalipon A.. 1993; Characterization of B-cell epitopes on IpaB, an invasion-associated antigen of Shigella flexneri: identification of an immunodominant domain recognized during natural infection. Infect Immun61:3825–3831
    [Google Scholar]
  10. Björnfot A. C., Lavander M., Forsberg A., Wolf-Watz H.. 2009; Autoproteolysis of YscU of Yersinia pseudotuberculosis is important for regulation of expression and secretion of Yop proteins. J Bacteriol191:4259–4267
    [Google Scholar]
  11. Blocker A., Jouihri N., Larquet E., Gounon P., Ebel F., Parsot C., Sansonetti P. J., Allaoui A.. 2001; Structure and composition of the Shigella flexneri “needle complex”, a part of its type III secreton. Mol Microbiol39:652–663
    [Google Scholar]
  12. Blocker A., Komoriya K., Aizawa S.. 2003; Type III secretion systems and bacterial flagella: insights into their function from structural similarities. Proc Natl Acad Sci U S A100:3027–3030
    [Google Scholar]
  13. Botteaux A., Sani M., Kayath C. A., Boekema E. J., Allaoui A.. 2008; Spa32 interaction with the inner-membrane Spa40 component of the type III secretion system of Shigella flexneri is required for the control of the needle length by a molecular tape measure mechanism. Mol Microbiol70:1515–1528
    [Google Scholar]
  14. Botteaux A., Sory M. P., Biskri L., Parsot C., Allaoui A.. 2009; MxiC is secreted by and controls the substrate specificity of the Shigella flexneri type III secretion apparatus. Mol Microbiol71:449–460
    [Google Scholar]
  15. Dale C., Young S. A., Haydon D. T., Welburn S. C.. 2001; The insect endosymbiont Sodalis glossinidius utilizes a type III secretion system for cell invasion. Proc Natl Acad Sci U S A98:1883–1888
    [Google Scholar]
  16. Dale C., Plague G. R., Wang B., Ochman H., Moran N. A.. 2002; Type III secretion systems and the evolution of mutualistic endosymbiosis. Proc Natl Acad Sci U S A99:12397–12402
    [Google Scholar]
  17. Deane J. E., Graham S. C., Mitchell E. P., Flot D., Johnson S., Lea S. M.. 2008; Crystal structure of Spa40, the specificity switch for the Shigella flexneri type III secretion system. Mol Microbiol69:267–276
    [Google Scholar]
  18. Edqvist P. J., Olsson J., Lavander M., Sundberg L., Forsberg A., Wolf-Watz H., Lloyd S. A.. 2003; YscP and YscU regulate substrate specificity of the Yersinia type III secretion system. J Bacteriol185:2259–2266
    [Google Scholar]
  19. Ferris H. U., Furukawa Y., Minamino T., Kroetz M. B., Kihara M., Namba K., Macnab R. M.. 2005; FlhB regulates ordered export of flagellar components via autocleavage mechanism. J Biol Chem280:41236–41242
    [Google Scholar]
  20. Fraser G. M., Hirano T., Ferris H. U., Devgan L. L., Kihara M., Macnab R. M.. 2003; Substrate specificity of type III flagellar protein export in Salmonella is controlled by subdomain interactions in FlhB. Mol Microbiol48:1043–1057
    [Google Scholar]
  21. Jouihri N., Sory M. P., Page A. L., Gounon P., Parsot C., Allaoui A.. 2003; MxiK and MxiN interact with the Spa47 ATPase and are required for transit of the needle components MxiH and MxiI, but not of Ipa proteins, through the type III secretion apparatus of Shigella flexneri. Mol Microbiol49:755–767
    [Google Scholar]
  22. Journet L., Agrain C., Broz P., Cornelis G. R.. 2003; The needle length of bacterial injectisomes is determined by a molecular ruler. Science302:1757–1760
    [Google Scholar]
  23. Kane C. D., Schuch R., Day W. A. Jr, Maurelli A. T.. 2002; MxiE regulates intracellular expression of factors secreted by the Shigella flexneri 2a type III secretion system. J Bacteriol184:4409–4419
    [Google Scholar]
  24. Kubori T., Sukhan A., Aizawa S. I., Galan J. E.. 2000; Molecular characterization and assembly of the needle complex of the Salmonella typhimurium type III protein secretion system. Proc Natl Acad Sci U S A97:10225–10230
    [Google Scholar]
  25. Kutsukake K., Minamino T., Yokoseki T.. 1994; Isolation and characterization of FliK-independent flagellation mutants from Salmonella typhimurium. J Bacteriol176:7625–7629
    [Google Scholar]
  26. Macnab R. M.. 1999; The bacterial flagellum: reversible rotary propellor and type III export apparatus. J Bacteriol181:7149–7153
    [Google Scholar]
  27. Macnab R. M.. 2004; Type III flagellar protein export and flagellar assembly. Biochim Biophys Acta1694:207–217
    [Google Scholar]
  28. Magdalena J., Hachani A., Chamekh M., Jouihri N., Gounon P., Blocker A., Allaoui A.. 2002; Spa32 regulates a switch in substrate specificity of the type III secreton of Shigella flexneri from needle components to Ipa proteins. J Bacteriol184:3433–3441
    [Google Scholar]
  29. Marlovits T. C., Kubori T., Sukhan A., Thomas D. R., Galan J. E., Unger V. M.. 2004; Structural insights into the assembly of the type III secretion needle complex. Science306:1040–1042
    [Google Scholar]
  30. Marlovits T. C., Kubori T., Lara-Tejero M., Thomas D., Unger V. M., Galán J. E.. 2006; Assembly of the inner rod determines needle length in the type III secretion injectisome. Nature441:637–640
    [Google Scholar]
  31. Martinez E., Bartolome B., de la Cruz F.. 1988; pACYC184-derived cloning vectors containing the multiple cloning site and lacZ alpha reporter gene of pUC8/9 and pUC18/19 plasmids. Gene68:159–162
    [Google Scholar]
  32. Menard R., Sansonetti P. J., Parsot C.. 1993; Nonpolar mutagenesis of the ipa genes defines IpaB, IpaC, and IpaD as effectors of Shigella flexneri entry into epithelial cells. J Bacteriol175:5899–5906
    [Google Scholar]
  33. Minamino T., Macnab R. M.. 2000; Domain structure of Salmonella FlhB, a flagellar export component responsible for substrate specificity switching. J Bacteriol182:4906–4914
    [Google Scholar]
  34. Minamino T., Namba K.. 2008; Distinct roles of the FliI ATPase and proton motive force in bacterial flagellar protein export. Nature451:485–488
    [Google Scholar]
  35. Morita-Ishihara T., Ogawa M., Sagara H., Yoshida M., Katayama E., Sasakawa C.. 2005; Shigella Spa33 is an essential C-ring component of type III secretion machinery. J Biol Chem281:599–607
    [Google Scholar]
  36. Page A. L., Fromont-Racine M., Sansonetti P., Legrain P., Parsot C.. 2001; Characterization of the interaction partners of secreted proteins and chaperones of Shigella flexneri. Mol Microbiol42:1133–1145
    [Google Scholar]
  37. Parsot C.. 2009; Shigella type III secretion effectors: how, where, when, for what purposes?. Curr Opin Microbiol12:110–116
    [Google Scholar]
  38. Penno C., Hachani A., Biskri L., Sansonetti P., Allaoui A., Parsot C.. 2006; Transcriptional slippage controls production of type III secretion apparatus components in Shigella flexneri. Mol Microbiol62:1460–1468
    [Google Scholar]
  39. Phalipon A., Arondel J., Nato F., Rouyre S., Mazie J. C., Sansonetti P. J.. 1992; Identification and characterization of B-cell epitopes of IpaC, an invasion-associated protein of Shigella flexneri. Infect Immun60:1919–1926
    [Google Scholar]
  40. Pilgram G. S., Engelsma-van Pelt A. M., Oostergetel G. T., Koerten H. K., Bouwstra J. A.. 1998; Study on the lipid organization of stratum corneum lipid models by (cryo-) electron diffraction. J Lipid Res39:1669–1676
    [Google Scholar]
  41. Riordan K. E., Schneewind O.. 2008; YscU cleavage and the assembly of Yersinia type III secretion machine complexes. Mol Microbiol68:1485–1501
    [Google Scholar]
  42. Sani M., Allaoui A., Fusetti F., Oostergetel G. T., Keegstra W., Boekema E. J.. 2007; Structural organization of the needle complex of the type III secretion apparatus of Shigella flexneri. Micron38:291–301
    [Google Scholar]
  43. Sansonetti P. J., d'Hauteville H., Formal S. B., Toucas M.. 1982; Plasmid-mediated invasiveness of “Shigella-like” Escherichia coli. Ann Microbiol (Paris133:351–355
    [Google Scholar]
  44. Sasakawa C., Komatsu K., Tobe T., Suzuki T., Yoshikawa M.. 1993; Eight genes in region 5 that form an operon are essential for invasion of epithelial cells by Shigella flexneri 2a. J Bacteriol175:2334–2346
    [Google Scholar]
  45. Schuch R., Maurelli A. T.. 2001; Spa33, a cell surface-associated subunit of the Mxi-Spa type III secretory pathway of Shigella flexneri, regulates Ipa protein traffic. Infect Immun69:2180–2189
    [Google Scholar]
  46. Tran Van Nhieu G., Ben-Ze'ev A., Sansonetti P. J.. 1997; Modulation of bacterial entry into epithelial cells by association between vinculin and the Shigella IpaA invasin. EMBO J16:2717–2729
    [Google Scholar]
  47. Viprey V., Del Greco A., Golinowski W., Broughton W. J., Perret X.. 1998; Symbiotic implications of type III protein secretion machinery in Rhizobium. Mol Microbiol28:1381–1389
    [Google Scholar]
  48. Wiesand U., Sorg I., Amstutz M., Wagner S., Van den Heuvel J., Lührs T., Cornelis G. R., Heinz D. W.. 2009; Structure of the type III secretion recognition protein YscU from Yersinia enterocolitica. J Mol Biol385:854–866
    [Google Scholar]
  49. Williams A. W., Yamaguchi S., Togashi F., Aizawa S. I., Kawagishi I., Macnab R. M.. 1996; Mutations in fliK and flhB affecting flagellar hook and filament assembly in Salmonella typhimurium. J Bacteriol178:2960–2970
    [Google Scholar]
  50. Wood S. E., Jin J., Lloyd S. A.. 2008; YscP and YscU switch the substrate specificity of the Yersinia type III secretion system by regulating export of the inner rod protein YscI. J Bacteriol190:4252–4262
    [Google Scholar]
  51. Zarivach R., Deng W., Vuckovic M., Felise H. B., Nguyen H. V., Miller S. I., Finlay B. B., Strynadka N. C.. 2008; Structural analysis of the essential self-cleaving type III secretion proteins EscU and SpaS. Nature453:124–127
    [Google Scholar]
  52. Zenk S. F., Stabat D., Hodgkinson J. L., Veenendaal A. K., Johnson S., Blocker A. J.. 2007; Identification of minor inner-membrane components of the Shigella type III secretion system ‘needle complex’. Microbiology153:2405–2415
    [Google Scholar]
  53. Zhu K., Gonzalez-Pedrajo B., Macnab R. M.. 2002; Interactions among membrane and soluble components of the flagellar export apparatus of Salmonella. Biochemistry41:9516–9524
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.039651-0
Loading
/content/journal/micro/10.1099/mic.0.039651-0
Loading

Data & Media loading...

Most cited this month Most Cited RSS feed

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