1887

Abstract

The type III secretion apparatus (T3SA), which is evolutionarily and structurally related to the bacterial flagellar hook basal body, is a key virulence factor used by many Gram-negative bacteria to inject effector proteins into host cells. A hollow extracellular needle forms the injection conduit of the T3SA. Its length is tightly controlled to match specific structures at the bacterial and host-cell surfaces but how this occurs remains incompletely understood. The needle is topped by a tip complex, which senses the host cell and inserts as a translocation pore in the host membrane when secretion is activated. The interaction of two conserved proteins, inner-membrane Spa40 and secreted Spa32, respectively, in , is proposed to regulate needle length and to flick a type III secretion substrate specificity switch from needle components/Spa32 to translocator/effector substrates. We found that, as in T3SAs from other species, substitution N257A within the conserved cytoplasmic NPTH region in Spa40 prevented its autocleavage and substrate specificity switching. Yet, the mutant made only slightly longer needles with a few needle tip complexes, although it could not form translocation pores. On the other hand, Δ, which makes extremely long needles and also formed only few tip complexes, could still form some translocation pores, indicating that it could switch substrate specificity to some extent. Therefore, loss of needle length control and defects in secretion specificity switching are not tightly coupled in either a Δ mutant or a mutant.

  • This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.059618-0
2012-07-01
2024-03-28
Loading full text...

Full text loading...

/deliver/fulltext/micro/158/7/1884.html?itemId=/content/journal/micro/10.1099/mic.0.059618-0&mimeType=html&fmt=ahah

References

  1. Agrain C., Callebaut I., Journet L., Sorg I., Paroz C., Mota L. J., Cornelis G. R. ( 2005). Characterization of a Type III secretion substrate specificity switch (T3S4) domain in YscP from Yersinia enterocolitica . Mol Microbiol 56:54–67 [View Article][PubMed]
    [Google Scholar]
  2. Allaoui A., Sansonetti P. J., Parsot C. ( 1992). MxiJ, a lipoprotein involved in secretion of Shigella Ipa invasins, is homologous to YscJ, a secretion factor of the Yersinia Yop proteins. J Bacteriol 174:7661–7669[PubMed]
    [Google Scholar]
  3. Allaoui A., Woestyn S., Sluiters C., Cornelis G. R. ( 1994). YscU, a Yersinia enterocolitica inner membrane protein involved in Yop secretion. J Bacteriol 176:4534–4542[PubMed]
    [Google Scholar]
  4. Allaoui A., Sansonetti P. J., Ménard 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 Microbiol 17:461–470 [View Article][PubMed]
    [Google Scholar]
  5. Bahrani F. K., Sansonetti P. J., Parsot C. ( 1997). Secretion of Ipa proteins by Shigella flexneri: inducer molecules and kinetics of activation. Infect Immun 65:4005–4010[PubMed]
    [Google Scholar]
  6. Barzu S., Nato F., Rouyre S., Mazie J. C., Sansonetti P., 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 Immun 61:3825–3831[PubMed]
    [Google Scholar]
  7. 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 Bacteriol 191:4259–4267 [View Article][PubMed]
    [Google Scholar]
  8. Blocker A., Gounon P., Larquet E., Niebuhr K., Cabiaux V., Parsot C., Sansonetti P. ( 1999). The tripartite type III secreton of Shigella flexneri inserts IpaB and IpaC into host membranes. J Cell Biol 147:683–693 [View Article][PubMed]
    [Google Scholar]
  9. Blocker A., Jouihri N., Larquet E., Gounon P., Ebel F., Parsot C., Sansonetti P., Allaoui A. ( 2001). Structure and composition of the Shigella flexneri “needle complex”, a part of its type III secreton. Mol Microbiol 39:652–663 [View Article][PubMed]
    [Google Scholar]
  10. 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 A 100:3027–3030 [View Article][PubMed]
    [Google Scholar]
  11. 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 Microbiol 70:1515–1528 [View Article][PubMed]
    [Google Scholar]
  12. Botteaux A., Kayath C. A., Page A. L., Jouihri N., Sani M., Boekema E., Biskri L., Parsot C., Allaoui A. ( 2010). The 33 carboxyl-terminal residues of Spa40 orchestrate the multi-step assembly process of the type III secretion needle complex in Shigella flexneri . Microbiology 156:2807–2817 [View Article][PubMed]
    [Google Scholar]
  13. Buchrieser C., Glaser P., Rusniok C., Nedjari H., D’Hauteville H., Kunst F., Sansonetti P., Parsot C. ( 2000). The virulence plasmid pWR100 and the repertoire of proteins secreted by the type III secretion apparatus of Shigella flexneri . Mol Microbiol 38:760–771 [View Article][PubMed]
    [Google Scholar]
  14. Chevance F. F., Hughes K. T. ( 2008). Coordinating assembly of a bacterial macromolecular machine. Nat Rev Microbiol 6:455–465 [View Article][PubMed]
    [Google Scholar]
  15. Cornelis G. R. ( 2006). The type III secretion injectisome. Nat Rev Microbiol 4:811–825 [View Article][PubMed]
    [Google Scholar]
  16. Datsenko K. A., Wanner B. L. ( 2000). One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci U S A 97:6640–6645 [View Article][PubMed]
    [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 Microbiol 69:267–276 [View Article][PubMed]
    [Google Scholar]
  18. Dykxhoorn D. M., St Pierre R., Linn T. ( 1996). A set of compatible tac promoter expression vectors. Gene 177:133–136 [View Article][PubMed]
    [Google Scholar]
  19. 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 Bacteriol 185:2259–2266 [View Article][PubMed]
    [Google Scholar]
  20. Erhardt M., Singer H. M., Wee D. H., Keener J. P., Hughes K. T. ( 2011). An infrequent molecular ruler controls flagellar hook length in Salmonella enterica . EMBO J 30:2948–2961 [View Article][PubMed]
    [Google Scholar]
  21. Ferris H. U., Minamino T. ( 2006). Flipping the switch: bringing order to flagellar assembly. Trends Microbiol 14:519–526 [View Article][PubMed]
    [Google Scholar]
  22. 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 Chem 280:41236–41242 [View Article][PubMed]
    [Google Scholar]
  23. 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 Microbiol 48:1043–1057 [View Article][PubMed]
    [Google Scholar]
  24. Frye J., Karlinsey J. E., Felise H. R., Marzolf B., Dowidar N., McClelland M., Hughes K. T. ( 2006). Identification of new flagellar genes of Salmonella enterica serovar Typhimurium. J Bacteriol 188:2233–2243 [View Article][PubMed]
    [Google Scholar]
  25. Harlow E., Lane D. ( 1988). Storing and purifying antibodies. Immunoaffinity purification of antibodies. Antibodies: a Laboratory Manual312–318 Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  26. Hirano T., Yamaguchi S., Oosawa K., Aizawa S. ( 1994). Roles of FliK and FlhB in determination of flagellar hook length in Salmonella typhimurium . J Bacteriol 176:5439–5449[PubMed]
    [Google Scholar]
  27. Journet L., Agrain C., Broz P., Cornelis G. R. ( 2003). The needle length of bacterial injectisomes is determined by a molecular ruler. Science 302:1757–1760 [View Article][PubMed]
    [Google Scholar]
  28. Kenjale R., Wilson J., Zenk S. F., Saurya S., Picking W. L., Picking W. D., Blocker A. ( 2005). The needle component of the type III secreton of Shigella regulates the activity of the secretion apparatus. J Biol Chem 280:42929–42937 [View Article][PubMed]
    [Google Scholar]
  29. Kotloff K. L., Winickoff J. P., Ivanoff B., Clemens J. D., Swerdlow D. L., Sansonetti P. J., Adak G. K., Levine M. M. ( 1999). Global burden of Shigella infections: implications for vaccine development and implementation of control strategies. Bull World Health Organ 77:651–666[PubMed]
    [Google Scholar]
  30. Kubori T., Matsushima Y., Nakamura D., Uralil J., Lara-Tejero M., Sukhan A., Galán J. E., Aizawa S. I. ( 1998). Supramolecular structure of the Salmonella typhimurium type III protein secretion system. Science 280:602–605 [View Article][PubMed]
    [Google Scholar]
  31. Lavander M., Sundberg L., Edqvist P. J., Lloyd S. A., Wolf-Watz H., Forsberg A. ( 2002). Proteolytic cleavage of the FlhB homologue YscU of Yersinia pseudotuberculosis is essential for bacterial survival but not for type III secretion. J Bacteriol 184:4500–4509 [View Article][PubMed]
    [Google Scholar]
  32. Le Gall T., Mavris M., Martino M. C., Bernardini M. L., Denamur E., Parsot C. ( 2005). Analysis of virulence plasmid gene expression defines three classes of effectors in the type III secretion system of Shigella flexneri . Microbiology 151:951–962 [View Article][PubMed]
    [Google Scholar]
  33. Lorenz C., Büttner D. ( 2011). Secretion of early and late substrates of the type III secretion system from Xanthomonas is controlled by HpaC and the C-terminal domain of HrcU. Mol Microbiol 79:447–467 [View Article][PubMed]
    [Google Scholar]
  34. Lountos G. T., Austin B. P., Nallamsetty S., Waugh D. S. ( 2009). Atomic resolution structure of the cytoplasmic domain of Yersinia pestis YscU, a regulatory switch involved in type III secretion. Protein Sci 18:467–474 [View Article][PubMed]
    [Google Scholar]
  35. 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 Bacteriol 184:3433–3441 [View Article][PubMed]
    [Google Scholar]
  36. Makishima S., Komoriya K., Yamaguchi S., Aizawa S. I. ( 2001). Length of the flagellar hook and the capacity of the type III export apparatus. Science 291:2411–2413 [View Article][PubMed]
    [Google Scholar]
  37. Marlovits T. C., Kubori T., Sukhan A., Thomas D. R., Galán J. E., Unger V. M. ( 2004). Structural insights into the assembly of the type III secretion needle complex. Science 306:1040–1042 [View Article][PubMed]
    [Google Scholar]
  38. 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. Nature 441:637–640 [View Article][PubMed]
    [Google Scholar]
  39. Martinez-Argudo I., Blocker A. J. ( 2010). The Shigella T3SS needle transmits a signal for MxiC release, which controls secretion of effectors. Mol Microbiol 78:1365–1378 [View Article][PubMed]
    [Google Scholar]
  40. Maurelli A. T., Blackmon B., Curtiss R. III ( 1984). Temperature-dependent expression of virulence genes in Shigella species. Infect Immun 43:195–201[PubMed]
    [Google Scholar]
  41. Meitert T., Pencu E., Ciudin L., Tonciu M., Mihai I., Nicolescu S. ( 1991). Correlation between Congo red binding as virulence marker in Shigella species and Sereny test. Roum Arch Microbiol Immunol 50:45–52[PubMed]
    [Google Scholar]
  42. Ménard 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 Bacteriol 175:5899–5906[PubMed]
    [Google Scholar]
  43. Minamino T., Macnab R. M. ( 2000). Domain structure of Salmonella FlhB, a flagellar export component responsible for substrate specificity switching. J Bacteriol 182:4906–4914 [View Article][PubMed]
    [Google Scholar]
  44. Minamino T., Saijo-Hamano Y., Furukawa Y., González-Pedrajo B., Macnab R. M., Namba K. ( 2004). Domain organization and function of Salmonella FliK, a flagellar hook-length control protein. J Mol Biol 341:491–502 [View Article][PubMed]
    [Google Scholar]
  45. Minamino T., Ferris H. U., Moriya N., Kihara M., Namba K. ( 2006). Two parts of the T3S4 domain of the hook-length control protein FliK are essential for the substrate specificity switching of the flagellar type III export apparatus. J Mol Biol 362:1148–1158 [View Article][PubMed]
    [Google Scholar]
  46. Minamino T., Imada K., Namba K. ( 2008). Mechanisms of type III protein export for bacterial flagellar assembly. Mol Biosyst 4:1105–1115 [View Article][PubMed]
    [Google Scholar]
  47. Minamino T., Moriya N., Hirano T., Hughes K. T., Namba K. ( 2009). Interaction of FliK with the bacterial flagellar hook is required for efficient export specificity switching. Mol Microbiol 74:239–251 [View Article][PubMed]
    [Google Scholar]
  48. Mizuno S., Amida H., Kobayashi N., Aizawa S., Tate S. ( 2011). The NMR structure of FliK, the trigger for the switch of substrate specificity in the flagellar type III secretion apparatus. J Mol Biol 409:558–573 [View Article][PubMed]
    [Google Scholar]
  49. Moriya N., Minamino T., Hughes K. T., Macnab R. M., Namba K. ( 2006). The type III flagellar export specificity switch is dependent on FliK ruler and a molecular clock. J Mol Biol 359:466–477 [View Article][PubMed]
    [Google Scholar]
  50. Morris D. P., Roush E. D., Thompson J. W., Moseley M. A., Murphy J. W., McMurry J. L. ( 2010). Kinetic characterization of Salmonella FliK–FlhB interactions demonstrates complexity of the Type III secretion substrate-specificity switch. Biochemistry 49:6386–6393 [View Article][PubMed]
    [Google Scholar]
  51. Mota L. J., Journet L., Sorg I., Agrain C., Cornelis G. R. ( 2005). Bacterial injectisomes: needle length does matter. Science 307:1278 [View Article][PubMed]
    [Google Scholar]
  52. Ohnishi K., Ohto Y., Aizawa S., Macnab R. M., Iino T. ( 1994). FlgD is a scaffolding protein needed for flagellar hook assembly in Salmonella typhimurium . J Bacteriol 176:2272–2281[PubMed]
    [Google Scholar]
  53. Parsot C. ( 2009). Shigella type III secretion effectors: how, where, when, for what purposes?. Curr Opin Microbiol 12:110–116 [View Article][PubMed]
    [Google Scholar]
  54. Parsot C., Ageron E., Penno C., Mavris M., Jamoussi K., d’Hauteville H., Sansonetti P., Demers B. ( 2005). A secreted anti-activator, OspD1, and its chaperone, Spa15, are involved in the control of transcription by the type III secretion apparatus activity in Shigella flexneri . Mol Microbiol 56:1627–1635 [View Article][PubMed]
    [Google Scholar]
  55. Patterson-Delafield J., Martinez R. J., Stocker B. A., Yamaguchi S. ( 1973). A new fla gene in Salmonella typhimuriumflaR – and its mutant phenotype-superhooks. Arch Mikrobiol 90:107–120 [View Article][PubMed]
    [Google Scholar]
  56. 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 Immun 60:1919–1926[PubMed]
    [Google Scholar]
  57. Poyraz O., Schmidt H., Seidel K., Delissen F., Ader C., Tenenboim H., Goosmann C., Laube B., Thünemann A. F. & other authors ( 2010). Protein refolding is required for assembly of the type three secretion needle. Nat Struct Mol Biol 17:788–792 [View Article][PubMed]
    [Google Scholar]
  58. Sansonetti P. J., Kopecko D. J., Formal S. B. ( 1982). Involvement of a plasmid in the invasive ability of Shigella flexneri . Infect Immun 35:852–860[PubMed]
    [Google Scholar]
  59. Schroeder G. N., Hilbi H. ( 2008). Molecular pathogenesis of Shigella spp.: controlling host cell signaling, invasion, and death by type III secretion. Clin Microbiol Rev 21:134–156 [View Article][PubMed]
    [Google Scholar]
  60. Shen D. K., Saurya S., Wagner C., Nishioka H., Blocker A. J. ( 2010). Domains of the Shigella flexneri type III secretion system IpaB protein involved in secretion regulation. Infect Immun 78:4999–5010 [View Article][PubMed]
    [Google Scholar]
  61. Shibata S., Takahashi N., Chevance F. F., Karlinsey J. E., Hughes K. T., Aizawa S. ( 2007). FliK regulates flagellar hook length as an internal ruler. Mol Microbiol 64:1404–1415 [View Article][PubMed]
    [Google Scholar]
  62. Sorg I., Wagner S., Amstutz M., Müller S. A., Broz P., Lussi Y., Engel A., Cornelis G. R. ( 2007). YscU recognizes translocators as export substrates of the Yersinia injectisome. EMBO J 26:3015–3024 [View Article][PubMed]
    [Google Scholar]
  63. Suzuki T., Iino T. ( 1981). Role of the flaR gene in flagellar hook formation in Salmonella spp. J Bacteriol 148:973–979[PubMed]
    [Google Scholar]
  64. Tamano K., Aizawa S., Katayama E., Nonaka T., Imajoh-Ohmi S., Kuwae A., Nagai S., Sasakawa C. ( 2000). Supramolecular structure of the Shigella type III secretion machinery: the needle part is changeable in length and essential for delivery of effectors. EMBO J 19:3876–3887 [View Article][PubMed]
    [Google Scholar]
  65. Tamano K., Katayama E., Toyotome T., Sasakawa C. ( 2002). Shigella Spa32 is an essential secretory protein for functional type III secretion machinery and uniformity of its needle length. J Bacteriol 184:1244–1252 [View Article][PubMed]
    [Google Scholar]
  66. Veenendaal A. K., Hodgkinson J. L., Schwarzer L., Stabat D., Zenk S. F., Blocker A. J. ( 2007). The type III secretion system needle tip complex mediates host cell sensing and translocon insertion. Mol Microbiol 63:1719–1730 [View Article][PubMed]
    [Google Scholar]
  67. Wagner S., Stenta M., Metzger L. C., Dal Peraro M., Cornelis G. R. ( 2010). Length control of the injectisome needle requires only one molecule of Yop secretion protein P (YscP). Proc Natl Acad Sci U S A 107:13860–13865 [View Article][PubMed]
    [Google Scholar]
  68. West N. P., Sansonetti P., Mounier J., Exley R. M., Parsot C., Guadagnini S., Prévost M. C., Prochnicka-Chalufour A., Delepierre M. & other authors ( 2005). Optimization of virulence functions through glucosylation of Shigella LPS. Science 307:1313–1317 [View Article][PubMed]
    [Google Scholar]
  69. 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 Biol 385:854–866 [View Article][PubMed]
    [Google Scholar]
  70. 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. Nature 453:124–127 [View Article][PubMed]
    [Google Scholar]
  71. 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’. Microbiology 153:2405–2415 [View Article][PubMed]
    [Google Scholar]
  72. Zhu K., González-Pedrajo B., Macnab R. M. ( 2002). Interactions among membrane and soluble components of the flagellar export apparatus of Salmonella . Biochemistry 41:9516–9524 [View Article][PubMed]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.059618-0
Loading
/content/journal/micro/10.1099/mic.0.059618-0
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