1887

Abstract

The majority of Gram-negative plant- and animal-pathogenic bacteria employ a type III secretion (T3S) system to deliver effector proteins to eukaryotic cells. Members of the YscU protein family are essential components of the T3S system and consist of a transmembrane and a cytoplasmic region that is autocatalytically cleaved at a conserved NPTH motif. YscU homologues interact with T3S substrate specificity switch (T3S4) proteins that alter the substrate specificity of the T3S system after assembly of the secretion apparatus. We previously showed that the YscU homologue HrcU from the plant pathogen pv. interacts with the T3S4 protein HpaC and is required for the secretion of translocon and effector proteins. In the present study, analysis of HrcU deletion, insertion and point mutant derivatives led to the identification of amino acid residues in the cytoplasmic region of HrcU (HrcU) that control T3S and translocation of the predicted inner rod protein HrpB2, the translocon protein HrpF and the effector protein AvrBs3. Mutations in the vicinity of the NPTH motif interfered with HrcU cleavage and/or the interaction of HrcU with HrpB2 and the T3S4 protein HpaC. However, HrcU function was not completely abolished, suggesting that HrcU cleavage is not crucial for pathogenicity and T3S. Given that mutations in HrcU differentially affected T3S and translocation of HrpB2 and effector proteins, we propose that HrcU controls the secretion of different T3S substrate classes by independent mechanisms.

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2014-03-01
2020-01-25
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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 Microbiol56:54–67 [CrossRef][PubMed]
    [Google Scholar]
  2. Ausubel F. M., Brent R., Kingston R. E., Moore D. D., Seidman J. G., Smith J. A., Struhl K..(editors) ( 1996;). Current Protocols in Molecular Biology New York: Wiley;
    [Google Scholar]
  3. Berger C., Robin G. P., Bonas U., Koebnik R..( 2010;). Membrane topology of conserved components of the type III secretion system from the plant pathogen Xanthomonas campestris pv. vesicatoria. Microbiology156:1963–1974 [CrossRef][PubMed]
    [Google Scholar]
  4. Bertani G..( 1951;). Studies on lysogenesis. I. The mode of phage liberation by lysogenic Escherichia coli. J Bacteriol62:293–300[PubMed]
    [Google Scholar]
  5. 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 [CrossRef][PubMed]
    [Google Scholar]
  6. 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 Microbiol39:652–663 [CrossRef][PubMed]
    [Google Scholar]
  7. Bolchi A., Ottonello S., Petrucco S..( 2005;). A general one-step method for the cloning of PCR products. Biotechnol Appl Biochem42:205–209 [CrossRef][PubMed]
    [Google Scholar]
  8. Büttner D..( 2012;). Protein export according to schedule: architecture, assembly, and regulation of type III secretion systems from plant- and animal-pathogenic bacteria. Microbiol Mol Biol Rev76:262–310 [CrossRef][PubMed]
    [Google Scholar]
  9. Büttner D., Bonas U..( 2010;). Regulation and secretion of Xanthomonas virulence factors. FEMS Microbiol Rev34:107–133 [CrossRef][PubMed]
    [Google Scholar]
  10. Büttner D., Nennstiel D., Klüsener B., Bonas U..( 2002;). Functional analysis of HrpF, a putative type III translocon protein from Xanthomonas campestris pv. vesicatoria. J Bacteriol184:2389–2398 [CrossRef][PubMed]
    [Google Scholar]
  11. Büttner D., Gürlebeck D., Noël L. D., Bonas U..( 2004;). HpaB from Xanthomonas campestris pv. vesicatoria acts as an exit control protein in type III-dependent protein secretion. Mol Microbiol54:755–768 [CrossRef][PubMed]
    [Google Scholar]
  12. Büttner D., Lorenz C., Weber E., Bonas U..( 2006;). Targeting of two effector protein classes to the type III secretion system by a HpaC- and HpaB-dependent protein complex from Xanthomonas campestris pv. vesicatoria. Mol Microbiol59:513–527 [CrossRef][PubMed]
    [Google Scholar]
  13. Cornelis G. R., Agrain C., Sorg I..( 2006;). Length control of extended protein structures in bacteria and bacteriophages. Curr Opin Microbiol9:201–206 [CrossRef][PubMed]
    [Google Scholar]
  14. Dangl J. L., Jones J. D. G..( 2001;). Plant pathogens and integrated defence responses to infection. Nature411:826–833 [CrossRef][PubMed]
    [Google Scholar]
  15. Daniels M. J., Barber C. E., Turner P. C., Sawczyc M. K., Byrde R. J. W., Fielding A. H..( 1984;). Cloning of genes involved in pathogenicity of Xanthomonas campestris pv. campestris using the broad host range cosmid pLAFR1. EMBO J3:3323–3328[PubMed]
    [Google Scholar]
  16. 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 [CrossRef][PubMed]
    [Google Scholar]
  17. Deane J. E., Abrusci P., Johnson S., Lea S. M..( 2010;). Timing is everything: the regulation of type III secretion. Cell Mol Life Sci67:1065–1075 [CrossRef][PubMed]
    [Google Scholar]
  18. DePamphilis M. L., Adler J..( 1971;). Fine structure and isolation of the hook-basal body complex of flagella from Escherichia coli and Bacillus subtilis. J Bacteriol105:384–395[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 Bacteriol185:2259–2266 [CrossRef][PubMed]
    [Google Scholar]
  20. Engler C., Kandzia R., Marillonnet S..( 2008;). A one pot, one step, precision cloning method with high throughput capability. PLoS ONE3:e3647 [CrossRef][PubMed]
    [Google Scholar]
  21. Escolar L., Van Den Ackerveken G., Pieplow S., Rossier O., Bonas U..( 2001;). Type III secretion and in planta recognition of the Xanthomonas avirulence proteins AvrBs1 and AvrBsT. Mol Plant Pathol2:287–296 [CrossRef][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 Chem280:41236–41242 [CrossRef][PubMed]
    [Google Scholar]
  23. Figurski D. H., Helinski D. R..( 1979;). Replication of an origin-containing derivative of plasmid RK2 dependent on a plasmid function provided in trans. Proc Natl Acad Sci U S A76:1648–1652 [CrossRef][PubMed]
    [Google Scholar]
  24. Francis N. R., Sosinsky G. E., Thomas D., DeRosier D. J..( 1994;). Isolation, characterization and structure of bacterial flagellar motors containing the switch complex. J Mol Biol235:1261–1270 [CrossRef][PubMed]
    [Google Scholar]
  25. 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 [CrossRef][PubMed]
    [Google Scholar]
  26. Ghosh P..( 2004;). Process of protein transport by the type III secretion system. Microbiol Mol Biol Rev68:771–795 [CrossRef][PubMed]
    [Google Scholar]
  27. Hartmann N., Schulz S., Lorenz C., Fraas S., Hause G., Büttner D..( 2012;). Characterization of HrpB2 from Xanthomonas campestris pv. vesicatoria identifies protein regions that are essential for type III secretion pilus formation. Microbiology158:1334–1349 [CrossRef][PubMed]
    [Google Scholar]
  28. He S. Y., Nomura K., Whittam T. S..( 2004;). Type III protein secretion mechanism in mammalian and plant pathogens. Biochim Biophys Acta1694:181–206 [CrossRef][PubMed]
    [Google Scholar]
  29. Jones J. B., Lacy G. H., Bouzar H., Stall R. E., Schaad N. W..( 2004;). Reclassification of the xanthomonads associated with bacterial spot disease of tomato and pepper. Syst Appl Microbiol27:755–762 [CrossRef][PubMed]
    [Google Scholar]
  30. Knoop V., Staskawicz B., Bonas U..( 1991;). Expression of the avirulence gene avrBs3 from Xanthomonas campestris pv. vesicatoria is not under the control of hrp genes and is independent of plant factors. J Bacteriol173:7142–7150[PubMed]
    [Google Scholar]
  31. Kousik C. S., Ritchie D. F..( 1998;). Response of bell pepper cultivars to bacterial spot pathogen races that individually overcome major resistance genes. Plant Dis82:181–186 [CrossRef]
    [Google Scholar]
  32. 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. Science280:602–605 [CrossRef][PubMed]
    [Google Scholar]
  33. Kutsukake K., Minamino T., Yokoseki T..( 1994;). Isolation and characterization of FliK-independent flagellation mutants from Salmonella typhimurium. J Bacteriol176:7625–7629[PubMed]
    [Google Scholar]
  34. 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 Bacteriol184:4500–4509 [CrossRef][PubMed]
    [Google Scholar]
  35. Lorenz C..( 2009;). Functional characterization of the conserved components HrcN und HrcU of the type III secretion system from Xanthomonas campestris pv. vesicatoria PhD thesis, Martin-Luther-University Halle-Wittenberg; Halle, Germany:
    [Google Scholar]
  36. Lorenz C., Büttner D..( 2009;). Functional characterization of the type III secretion ATPase HrcN from the plant pathogen Xanthomonas campestris pv. vesicatoria. J Bacteriol191:1414–1428 [CrossRef][PubMed]
    [Google Scholar]
  37. 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 Microbiol79:447–467 [CrossRef][PubMed]
    [Google Scholar]
  38. Lorenz C., Schulz S., Wolsch T., Rossier O., Bonas U., Büttner D..( 2008;). HpaC controls substrate specificity of the Xanthomonas type III secretion system. PLoS Pathog4:e1000094 [CrossRef][PubMed]
    [Google Scholar]
  39. Lorenz C., Hausner J., Büttner D..( 2012;). HrcQ provides a docking site for early and late type III secretion substrates from Xanthomonas. PLoS ONE7:e51063 [CrossRef][PubMed]
    [Google Scholar]
  40. 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 Sci18:467–474 [CrossRef][PubMed]
    [Google Scholar]
  41. 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. Science306:1040–1042 [CrossRef][PubMed]
    [Google Scholar]
  42. Matteï P. J., Faudry E., Job V., Izoré T., Attree I., Dessen A..( 2011;). Membrane targeting and pore formation by the type III secretion system translocon. FEBS J278:414–426 [CrossRef][PubMed]
    [Google Scholar]
  43. Miao E. A., Mao D. P., Yudkovsky N., Bonneau R., Lorang C. G., Warren S. E., Leaf I. A., Aderem A..( 2010;). Innate immune detection of the type III secretion apparatus through the NLRC4 inflammasome. Proc Natl Acad Sci U S A107:3076–3080 [CrossRef][PubMed]
    [Google Scholar]
  44. Minsavage G. V., Dahlbeck D., Whalen M. C., Kearny B., Bonas U., Staskawicz B. J., Stall R. E..( 1990;). Gene-for-gene relationships specifying disease resistance in Xanthomonas campestris pv. vesicatoria – pepper interactions. Mol Plant Microbe Interact3:41–47 [CrossRef]
    [Google Scholar]
  45. Morbitzer R., Elsaesser J., Hausner J., Lahaye T..( 2011;). Assembly of custom TALE-type DNA binding domains by modular cloning. Nucleic Acids Res39:5790–5799 [CrossRef][PubMed]
    [Google Scholar]
  46. Mueller C. A., Broz P., Cornelis G. R..( 2008;). The type III secretion system tip complex and translocon. Mol Microbiol68:1085–1095 [CrossRef][PubMed]
    [Google Scholar]
  47. Noël L., Thieme F., Nennstiel D., Bonas U..( 2001;). cDNA-AFLP analysis unravels a genome-wide hrpG-regulon in the plant pathogen Xanthomonas campestris pv. vesicatoria. Mol Microbiol41:1271–1281 [CrossRef][PubMed]
    [Google Scholar]
  48. Noël L., Thieme F., Gäbler J., Büttner D., Bonas U..( 2003;). XopC and XopJ, two novel type III effector proteins from Xanthomonas campestris pv. vesicatoria. J Bacteriol185:7092–7102 [CrossRef][PubMed]
    [Google Scholar]
  49. Römer P., Strauss T., Hahn S., Scholze H., Morbitzer R., Grau J., Bonas U., Lahaye T..( 2009;). Recognition of AvrBs3-like proteins is mediated by specific binding to promoters of matching pepper Bs3 alleles. Plant Physiol150:1697–1712 [CrossRef][PubMed]
    [Google Scholar]
  50. Ronald P. C., Staskawicz B. J..( 1988;). The avirulence gene avrBs1 from Xanthomonas campestris pv. vesicatoria encodes a 50-kD protein. Mol Plant Microbe Interact1:191–198 [CrossRef][PubMed]
    [Google Scholar]
  51. Rossier O., Wengelnik K., Hahn K., Bonas U..( 1999;). The Xanthomonas Hrp type III system secretes proteins from plant and mammalian bacterial pathogens. Proc Natl Acad Sci U S A96:9368–9373 [CrossRef][PubMed]
    [Google Scholar]
  52. Rossier O., Van den Ackerveken G., Bonas U..( 2000;). HrpB2 and HrpF from Xanthomonas are type III-secreted proteins and essential for pathogenicity and recognition by the host plant. Mol Microbiol38:828–838 [CrossRef][PubMed]
    [Google Scholar]
  53. 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 [CrossRef][PubMed]
    [Google Scholar]
  54. Smith T. G., Pereira L., Hoover T. R..( 2009;). Helicobacter pylori FlhB processing-deficient variants affect flagellar assembly but not flagellar gene expression. Microbiology155:1170–1180 [CrossRef][PubMed]
    [Google Scholar]
  55. 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 J26:3015–3024 [CrossRef][PubMed]
    [Google Scholar]
  56. Szczesny R., Jordan M., Schramm C., Schulz S., Cogez V., Bonas U., Büttner D..( 2010;). Functional characterization of the Xcs and Xps type II secretion systems from the plant pathogenic bacterium Xanthomonas campestris pv vesicatoria. New Phytol187:983–1002 [CrossRef][PubMed]
    [Google Scholar]
  57. Szurek B., Rossier O., Hause G., Bonas U..( 2002;). Type III-dependent translocation of the Xanthomonas AvrBs3 protein into the plant cell. Mol Microbiol46:13–23 [CrossRef][PubMed]
    [Google Scholar]
  58. Wengelnik K., Van den Ackerveken G., Bonas U..( 1996;). HrpG, a key hrp regulatory protein of Xanthomonas campestris pv. vesicatoria is homologous to two-component response regulators. Mol Plant Microbe Interact9:704–712 [CrossRef][PubMed]
    [Google Scholar]
  59. Wengelnik K., Rossier O., Bonas U..( 1999;). Mutations in the regulatory gene hrpG of Xanthomonas campestris pv. vesicatoria result in constitutive expression of all hrp genes. J Bacteriol181:6828–6831[PubMed]
    [Google Scholar]
  60. 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 [CrossRef][PubMed]
    [Google Scholar]
  61. 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[PubMed]
    [Google Scholar]
  62. 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 [CrossRef][PubMed]
    [Google Scholar]
  63. 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 [CrossRef][PubMed]
    [Google Scholar]
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