1887

Abstract

pandemic serotype O3 : K6 causes acute gastroenteritis, wound infections and septicaemia in humans. This organism encodes two type III secretion systems (T3SS1 and T3SS2); host-cell cytotoxicity has been attributed to T3SS1. Synthesis and secretion of T3SS1 proteins is positively regulated by ExsA, which is presumptively regulated by the ExsCDE pathway, similar to . Herein we deleted the putative from and found constitutive expression of the T3SS1 in broth culture as expected. More importantly, however, in a cell culture model, the Δ strain was unable to induce cytotoxicity, as measured by release of lactate dehydrogenase (LDH), or autophagy, as measured by LC3 conversion. This is markedly different from , where deletion of has no effect on host-cell cytolysis. Swarming and cytoadhesion were reduced for the deletion mutant and could be recovered along with T3SS1-induced HeLa cell cytotoxicity by expression of in the Δ strain. Loss of adhesion and swarming motility was associated with the loss of flagella biogenesis in the -deficient strain. Mouse mortality was unaffected by the deletion of compared with a wild-type control, suggesting that additional adhesins are important for intoxication . Based on these data, we conclude that ExsE contributes to the negative regulation of T3SS1 and, in addition, contributes to regulation of an adherence phenotype that is requisite for translocation of effector proteins into HeLa cells.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.059931-0
2012-09-01
2024-03-29
Loading full text...

Full text loading...

/deliver/fulltext/micro/158/9/2303.html?itemId=/content/journal/micro/10.1099/mic.0.059931-0&mimeType=html&fmt=ahah

References

  1. Belas M. R., Colwell R. R. ( 1982a). Adsorption kinetics of laterally and polarly flagellated Vibrio . J Bacteriol 151:1568–1580[PubMed]
    [Google Scholar]
  2. Belas M. R., Colwell R. R. ( 1982b). Scanning electron microscope observation of the swarming phenomenon of Vibrio parahaemolyticus . J Bacteriol 150:956–959[PubMed]
    [Google Scholar]
  3. Bleves S., Marenne M. N., Detry G., Cornelis G. R. ( 2002). Up-regulation of the Yersinia enterocolitica yop regulon by deletion of the flagellum master operon flhDC . J Bacteriol 184:3214–3223 [View Article][PubMed]
    [Google Scholar]
  4. Broberg C. A., Zhang L., Gonzalez H., Laskowski-Arce M. A., Orth K. ( 2010). A Vibrio effector protein is an inositol phosphatase and disrupts host cell membrane integrity. Science 329:1660–1662 [View Article][PubMed]
    [Google Scholar]
  5. Burdette D. L., Yarbrough M. L., Orvedahl A., Gilpin C. J., Orth K. ( 2008). Vibrio parahaemolyticus orchestrates a multifaceted host cell infection by induction of autophagy, cell rounding, and then cell lysis. Proc Natl Acad Sci U S A 105:12497–12502 [View Article][PubMed]
    [Google Scholar]
  6. Burdette D. L., Seemann J., Orth K. ( 2009). Vibrio VopQ induces PI3-kinase-independent autophagy and antagonizes phagocytosis. Mol Microbiol 73:639–649 [View Article][PubMed]
    [Google Scholar]
  7. Casselli T., Lynch T., Southward C. M., Jones B. W., DeVinney R. ( 2008). Vibrio parahaemolyticus inhibition of Rho family GTPase activation requires a functional chromosome I type III secretion system. Infect Immun 76:2202–2211 [View Article][PubMed]
    [Google Scholar]
  8. Cisz M., Lee P. C., Rietsch A. ( 2008). ExoS controls the cell contact-mediated switch to effector secretion in Pseudomonas aeruginosa . J Bacteriol 190:2726–2738 [View Article][PubMed]
    [Google Scholar]
  9. Coburn B., Sekirov I., Finlay B. B. ( 2007). Type III secretion systems and disease. Clin Microbiol Rev 20:535–549 [View Article][PubMed]
    [Google Scholar]
  10. Daniels N. A., MacKinnon L., Bishop R., Altekruse S., Ray B., Hammond R. M., Thompson S., Wilson S., Bean N. H. & other authors ( 2000). Vibrio parahaemolyticus infections in the United States, 1973–1998. J Infect Dis 181:1661–1666 [View Article][PubMed]
    [Google Scholar]
  11. Dasgupta N., Lykken G. L., Wolfgang M. C., Yahr T. L. ( 2004). A novel anti-anti-activator mechanism regulates expression of the Pseudomonas aeruginosa type III secretion system. Mol Microbiol 53:297–308 [View Article][PubMed]
    [Google Scholar]
  12. Diaz M. R., King J. M., Yahr T. L. ( 2011). Intrinsic and extrinsic regulation of type III secretion gene expression in Pseudomonas aeruginosa . Front Microbiol 2:89[PubMed]
    [Google Scholar]
  13. Eichelberg K., Galán J. E. ( 2000). The flagellar sigma factor FliA (sigma28) regulates the expression of Salmonella genes associated with the centisome 63 type III secretion system. Infect Immun 68:2735–2743 [View Article][PubMed]
    [Google Scholar]
  14. Fälker S., Schmidt M. A., Heusipp G. ( 2005). DNA methylation in Yersinia enterocolitica: role of the DNA adenine methyltransferase in mismatch repair and regulation of virulence factors. Microbiology 151:2291–2299 [View Article][PubMed]
    [Google Scholar]
  15. Frank D. W. ( 1997). The exoenzyme S regulon of Pseudomonas aeruginosa . Mol Microbiol 26:621–629 [View Article][PubMed]
    [Google Scholar]
  16. Gode-Potratz C. J., Chodur D. M., McCarter L. L. ( 2010). Calcium and iron regulate swarming and type III secretion in Vibrio parahaemolyticus . J Bacteriol 192:6025–6038 [View Article][PubMed]
    [Google Scholar]
  17. Gode-Potratz C. J., Kustusch R. J., Breheny P. J., Weiss D. S., McCarter L. L. ( 2011). Surface sensing in Vibrio parahaemolyticus triggers a programme of gene expression that promotes colonization and virulence. Mol Microbiol 79:240–263 [View Article][PubMed]
    [Google Scholar]
  18. Hiyoshi H., Kodama T., Iida T., Honda T. ( 2010). Contribution of Vibrio parahaemolyticus virulence factors to cytotoxicity, enterotoxicity, and lethality in mice. Infect Immun 78:1772–1780 [View Article][PubMed]
    [Google Scholar]
  19. Hlady W. G., Klontz K. C. ( 1996). The epidemiology of Vibrio infections in Florida, 1981–1993. J Infect Dis 173:1176–1183 [View Article][PubMed]
    [Google Scholar]
  20. Hornef M. W., Roggenkamp A., Geiger A. M., Hogardt M., Jacobi C. A., Heesemann J. ( 2000). Triggering the ExoS regulon of Pseudomonas aeruginosa: A GFP-reporter analysis of exoenzyme (Exo)S, ExoT and ExoU synthesis. Microb Pathog 29:329–343 [View Article][PubMed]
    [Google Scholar]
  21. Hovey A. K., Frank D. W. ( 1995). Analyses of the DNA-binding and transcriptional activation properties of ExsA, the transcriptional activator of the Pseudomonas aeruginosa exoenzyme S regulon. J Bacteriol 177:4427–4436[PubMed]
    [Google Scholar]
  22. Iyoda S., Koizumi N., Satou H., Lu Y., Saitoh T., Ohnishi M., Watanabe H. ( 2006). The GrlR–GrlA regulatory system coordinately controls the expression of flagellar and LEE-encoded type III protein secretion systems in enterohemorrhagic Escherichia coli . J Bacteriol 188:5682–5692 [View Article][PubMed]
    [Google Scholar]
  23. Kabeya Y., Mizushima N., Ueno T., Yamamoto A., Kirisako T., Noda T., Kominami E., Ohsumi Y., Yoshimori T. ( 2000). LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing. EMBO J 19:5720–5728 [View Article][PubMed]
    [Google Scholar]
  24. Kodama T., Yamazaki C., Park K. S., Akeda Y., Iida T., Honda T. ( 2010). Transcription of Vibrio parahaemolyticus T3SS1 genes is regulated by a dual regulation system consisting of the ExsACDE regulatory cascade and H-NS. FEMS Microbiol Lett 311:10–17 [View Article][PubMed]
    [Google Scholar]
  25. Krachler A. M., Orth K. ( 2011). Functional characterization of the interaction between bacterial adhesin multivalent adhesion molecule 7 (MAM7) protein and its host cell ligands. J Biol Chem 286:38939–38947 [View Article][PubMed]
    [Google Scholar]
  26. Makino K., Oshima K., Kurokawa K., Yokoyama K., Uda T., Tagomori K., Iijima Y., Najima M., Nakano M. & other authors ( 2003). Genome sequence of Vibrio parahaemolyticus: a pathogenic mechanism distinct from that of V. cholerae . Lancet 361:743–749 [View Article][PubMed]
    [Google Scholar]
  27. Matlawska-Wasowska K., Finn R., Mustel A., O’Byrne C. P., Baird A. W., Coffey E. T., Boyd A. ( 2010). The Vibrio parahaemolyticus Type III Secretion Systems manipulate host cell MAPK for critical steps in pathogenesis. BMC Microbiol 10:329 [View Article][PubMed]
    [Google Scholar]
  28. McCarter L. L. ( 2004). Dual flagellar systems enable motility under different circumstances. J Mol Microbiol Biotechnol 7:18–29 [View Article][PubMed]
    [Google Scholar]
  29. McCaw M. L., Lykken G. L., Singh P. K., Yahr T. L. ( 2002). ExsD is a negative regulator of the Pseudomonas aeruginosa type III secretion regulon. Mol Microbiol 46:1123–1133 [View Article][PubMed]
    [Google Scholar]
  30. Mertens A., Nagler J., Hansen W., Gepts-Friedenreich E. ( 1979). Halophilic, lactose-positive Vibrio in a case of fatal septicemia. J Clin Microbiol 9:233–235[PubMed]
    [Google Scholar]
  31. Michiels T., Wattiau P., Brasseur R., Ruysschaert J. M., Cornelis G. ( 1990). Secretion of Yop proteins by Yersiniae . Infect Immun 58:2840–2849[PubMed]
    [Google Scholar]
  32. Milton D. L., Norqvist A., Wolf-Watz H. ( 1992). Cloning of a metalloprotease gene involved in the virulence mechanism of Vibrio anguillarum . J Bacteriol 174:7235–7244[PubMed]
    [Google Scholar]
  33. Milton D. L., O’Toole R., Horstedt P., Wolf-Watz H. ( 1996). Flagellin A is essential for the virulence of Vibrio anguillarum . J Bacteriol 178:1310–1319[PubMed]
    [Google Scholar]
  34. Morales V. M., Bäckman A., Bagdasarian M. ( 1991). A series of wide-host-range low-copy-number vectors that allow direct screening for recombinants. Gene 97:39–47 [View Article][PubMed]
    [Google Scholar]
  35. Morris J. G. Jr, Black R. E. ( 1985). Cholera and other vibrioses in the United States. N Engl J Med 312:343–350 [View Article][PubMed]
    [Google Scholar]
  36. Nishibuchi M., Fasano A., Russell R. G., Kaper J. B. ( 1992). Enterotoxigenicity of Vibrio parahaemolyticus with and without genes encoding thermostable direct hemolysin. Infect Immun 60:3539–3545[PubMed]
    [Google Scholar]
  37. Niu C., Graves J. D., Mokuolu F. O., Gilbert S. E., Gilbert E. S. ( 2005). Enhanced swarming of bacteria on agar plates containing the surfactant Tween 80. J Microbiol Methods 62:129–132 [View Article][PubMed]
    [Google Scholar]
  38. Ono T., Park K. S., Ueta M., Iida T., Honda T. ( 2006). Identification of proteins secreted via Vibrio parahaemolyticus type III secretion system 1. Infect Immun 74:1032–1042 [View Article][PubMed]
    [Google Scholar]
  39. Park K. S., Ono T., Rokuda M., Jang M. H., Okada K., Iida T., Honda T. ( 2004). Functional characterization of two type III secretion systems of Vibrio parahaemolyticus . Infect Immun 72:6659–6665 [View Article][PubMed]
    [Google Scholar]
  40. Park K. S., Arita M., Iida T., Honda T. ( 2005). vpaH, a gene encoding a novel histone-like nucleoid structure-like protein that was possibly horizontally acquired, regulates the biogenesis of lateral flagella in trh-positive Vibrio parahaemolyticus TH3996. Infect Immun 73:5754–5761 [View Article][PubMed]
    [Google Scholar]
  41. Pavankumar A. R., Ayyappasamy S. P., Sankaran K. ( 2012). Small RNA fragments in complex culture media cause alterations in protein profiles of three species of bacteria. Biotechniques 52:167–172[PubMed]
    [Google Scholar]
  42. Piñeyro P., Zhou X., Orfe L. H., Friel P. J., Lahmers K., Call D. R. ( 2010). Development of two animal models to study the function of Vibrio parahaemolyticus type III secretion systems. Infect Immun 78:4551–4559 [View Article][PubMed]
    [Google Scholar]
  43. Qadri F., Alam M. S., Nishibuchi M., Rahman T., Alam N. H., Chisti J., Kondo S., Sugiyama J., Bhuiyan N. A. & other authors ( 2003). Adaptive and inflammatory immune responses in patients infected with strains of Vibrio parahaemolyticus . J Infect Dis 187:1085–1096 [View Article][PubMed]
    [Google Scholar]
  44. Rietsch A., Vallet-Gely I., Dove S. L., Mekalanos J. J. ( 2005). ExsE, a secreted regulator of type III secretion genes in Pseudomonas aeruginosa . Proc Natl Acad Sci U S A 102:8006–8011 [View Article][PubMed]
    [Google Scholar]
  45. Ryan W. J. ( 1976). Marine vibrios associated with superficial septic lesions. J Clin Pathol 29:1014–1015 [View Article][PubMed]
    [Google Scholar]
  46. Salmond G. P., Reeves P. J. ( 1993). Membrane traffic wardens and protein secretion in gram-negative bacteria. Trends Biochem Sci 18:7–12 [View Article][PubMed]
    [Google Scholar]
  47. Sato H., Frank D. W. ( 2011). Multi-functional characteristics of the Pseudomonas aeruginosa type III needle-tip protein, PcrV; comparison to orthologs in other Gram-negative bacteria. Front Microbiol 2:142[PubMed] [CrossRef]
    [Google Scholar]
  48. Shinoda S., Okamoto K. ( 1977). Formation and function of Vibrio parahaemolyticus lateral flagella. J Bacteriol 129:1266–1271[PubMed]
    [Google Scholar]
  49. Soscia C., Hachani A., Bernadac A., Filloux A., Bleves S. ( 2007). Cross talk between type III secretion and flagellar assembly systems in Pseudomonas aeruginosa . J Bacteriol 189:3124–3132 [View Article][PubMed]
    [Google Scholar]
  50. Tison D. L., Kelly M. T. ( 1984). Vibrio species of medical importance. Diagn Microbiol Infect Dis 2:263–276 [View Article][PubMed]
    [Google Scholar]
  51. Troisfontaines P., Cornelis G. R. ( 2005). Type III secretion: more systems than you think. Physiology (Bethesda) 20:326–339 [View Article][PubMed]
    [Google Scholar]
  52. Urbanowski M. L., Brutinel E. D., Yahr T. L. ( 2007). Translocation of ExsE into Chinese hamster ovary cells is required for transcriptional induction of the Pseudomonas aeruginosa type III secretion system. Infect Immun 75:4432–4439 [View Article][PubMed]
    [Google Scholar]
  53. Vallis A. J., Yahr T. L., Barbieri J. T., Frank D. W. ( 1999). Regulation of ExoS production and secretion by Pseudomonas aeruginosa in response to tissue culture conditions. Infect Immun 67:914–920[PubMed]
    [Google Scholar]
  54. Xu M., Yamamoto K., Honda T., Xu M. ( 1994). Construction and characterization of an isogenic mutant of Vibrio parahaemolyticus having a deletion in the thermostable direct hemolysin-related hemolysin gene (trh). J Bacteriol 176:4757–4760[PubMed]
    [Google Scholar]
  55. Yahr T. L., Wolfgang M. C. ( 2006). Transcriptional regulation of the Pseudomonas aeruginosa type III secretion system. Mol Microbiol 62:631–640 [View Article][PubMed]
    [Google Scholar]
  56. Yarbrough M. L., Li Y., Kinch L. N., Grishin N. V., Ball H. L., Orth K. ( 2009). AMPylation of Rho GTPases by Vibrio VopS disrupts effector binding and downstream signaling. Science 323:269–272 [View Article][PubMed]
    [Google Scholar]
  57. Zhou X., Shah D. H., Konkel M. E., Call D. R. ( 2008). Type III secretion system 1 genes in Vibrio parahaemolyticus are positively regulated by ExsA and negatively regulated by ExsD. Mol Microbiol 69:747–764 [View Article][PubMed]
    [Google Scholar]
  58. Zhou X., Konkel M. E., Call D. R. ( 2009). Type III secretion system 1 of Vibrio parahaemolyticus induces oncosis in both epithelial and monocytic cell lines. Microbiology 155:837–851 [View Article][PubMed]
    [Google Scholar]
  59. Zhou X., Konkel M. E., Call D. R. ( 2010a). Regulation of type III secretion system 1 gene expression in Vibrio parahaemolyticus is dependent on interactions between ExsA, ExsC, and ExsD. Virulence 1:260–272 [View Article][PubMed]
    [Google Scholar]
  60. Zhou X., Konkel M. E., Call D. R. ( 2010b). Vp1659 is a Vibrio parahaemolyticus type III secretion system 1 protein that contributes to translocation of effector proteins needed to induce cytolysis, autophagy, and disruption of actin structure in HeLa cells. J Bacteriol 192:3491–3502 [View Article][PubMed]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.059931-0
Loading
/content/journal/micro/10.1099/mic.0.059931-0
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