1887

Abstract

is an important Gram-negative enteric pathogen affecting both animals and humans. It possesses a type III secretion system (T3SS) essential for pathogenesis. EseB, EseC and EseD have been shown to form a translocon complex after secretion, while EscC functions as a T3SS chaperone for EseB and EseD. In this paper we identify EscA, a protein required for accumulation and proper secretion of another translocon component, EseC. The gene is located upstream of and the EscA protein has the characteristics of T3SS chaperones. Cell fractionation experiments indicated that EscA is located in the cytoplasm and on the cytoplasmic membrane. Mutation with in-frame deletion of greatly decreased the secretion of EseC, while complementation of restored the wild-type secretion phenotype. The stabilization and accumulation of EseC in the cytoplasm were also affected in the absence of EscA. Mutation of did not affect the transcription of but reduced the accumulation level of EseC as measured by using an EseC-LacZ fusion protein in . Co-purification and co-immunoprecipitation studies demonstrated a specific interaction between EscA and EseC. Further analysis showed that residues 31–137 of EseC are required for EseC-EscA interaction. Mutation of EseC residues 31–137 reduced the secretion and accumulation of EseC in . Finally, infection experiments showed that mutations of EscA and residues 31–137 of EseC increased the LD by approximately 10-fold in blue gourami fish. These results indicated that EscA functions as a specific chaperone for EseC and contributes to the virulence of .

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.021865-0
2009-04-01
2020-07-13
Loading full text...

Full text loading...

/deliver/fulltext/micro/155/4/1260.html?itemId=/content/journal/micro/10.1099/mic.0.021865-0&mimeType=html&fmt=ahah

References

  1. Akeda Y., Galán J. E.. 2005; Chaperone release and unfolding of substrates in type III secretion. Nature437:911–915
    [Google Scholar]
  2. Beuzón C. R., Banks G., Deiwick J., Hensel M., Holden D. W.. 1999; pH-dependent secretion of SseB, a product of the SPI-2 type III secretion system of Salmonella typhimurium . Mol Microbiol33:806–816
    [Google Scholar]
  3. Bröms J. E., Forslund A. L., Forsberg A., Francis M. S.. 2003; PcrH of Pseudomonas aeruginosa is essential for secretion and assembly of the type III translocon. J Infect Dis188:1909–1921
    [Google Scholar]
  4. Bröms J. E., Edqvist P. J., Forsberg A., Francis M. S.. 2006; Tetratricopeptide repeats are essential for PcrH chaperone function in Pseudomonas aeruginosa type III secretion. FEMS Microbiol Lett256:57–66
    [Google Scholar]
  5. Büttner D., Bonas U.. 2002; Port of entry – the type III secretion translocon. Trends Microbiol10:186–192
    [Google Scholar]
  6. Büttner C. R., Sorg I., Cornelis G. R., Heinz D. W., Niemann H. H.. 2008; Structure of the Yersinia enterocolitica type III secretion translocator chaperone SycD. J Mol Biol375:997–1012
    [Google Scholar]
  7. Chen J. D., Lai S. Y., Huang S. L.. 1996; Molecular cloning, characterization, and sequencing of the hemolysin gene from Edwardsiella tarda . Arch Microbiol165:9–17
    [Google Scholar]
  8. Cook R. A., Tappe J. P.. 1985; Chronic enteritis associated with Edwardsiella tarda infection in Rockhopper penguins. J Am Vet Med Assoc187:1219–1220
    [Google Scholar]
  9. Cornelis G. R., Van Gijsegem F.. 2000; Assembly and function of type III secretory systems. Annu Rev Microbiol54:735–774
    [Google Scholar]
  10. Daniell S. J., Delahay R. M., Shaw R. K., Hartland E. L., Pallen M. J., Booy F., Ebel F., Knutton S., Frankel G.. 2001; Coiled-coil domain of enteropathogenic Escherichia coli type III secreted protein EspD is involved in EspA filament-mediated cell attachment and hemolysis. Infect Immun69:4055–4064
    [Google Scholar]
  11. Daniell S. J., Kocsis E., Morris E., Knutton S., Booy F. P., Frankel G.. 2003; 3D structure of EspA filaments from enteropathogenic Escherichia coli . Mol Microbiol49:301–308
    [Google Scholar]
  12. Darwin K. H., Miller V. L.. 2001; Type III secretion chaperone-dependent regulation: activation of virulence genes by SicA and InvF in Salmonella typhimurium . EMBO J20:1850–1862
    [Google Scholar]
  13. Delahay R. M., Frankel G.. 2002; Coiled-coil proteins associated with type III secretion systems: a versatile domain revisited. Mol Microbiol45:905–916
    [Google Scholar]
  14. Edqvist P. J., Bröms J. E., Betts H. J., Forsberg A., Pallen M. J., Francis M. S.. 2006; Tetratricopeptide repeats in the type-III-secretion chaperone, LcrH: their role in substrate binding and secretion. Mol Microbiol59:31–44
    [Google Scholar]
  15. Edwards R. A., Keller L. H., Schifferli D. M.. 1998; Improved allelic exchange vectors and their use to analyze 987P fimbria gene expression. Gene207:149–157
    [Google Scholar]
  16. Elliott S. J., Hutcheson S. W., Dubois M. S., Mellies J. L., Wainwright L. A., Batchelor M., Frankel G., Knutton S., Kaper J. B.. 1999; Identification of CesT, a chaperone for the type III secretion of Tir in enteropathogenic Escherichia coli . Mol Microbiol33:1176–1189
    [Google Scholar]
  17. Evans L. D., Stafford G. P., Ahmed S., Fraser G. M., Hughes C.. 2006; An escort mechanism for cycling of export chaperones during flagellum assembly. Proc Natl Acad Sci U S A103:17474–17479
    [Google Scholar]
  18. Francis M. S., Wolf-Watz H., Forsberg A.. 2002; Regulation of type III secretion systems. Curr Opin Microbiol5:166–172
    [Google Scholar]
  19. Gauthier A., Finlay B. B.. 2003; Translocated intimin receptor and its chaperone interact with ATPase of the type III secretion apparatus of enteropathogenic Escherichia coli . J Bacteriol185:6747–6755
    [Google Scholar]
  20. Ghosh P.. 2004; Process of protein transport by the type III secretion system. Microbiol Mol Biol Rev68:771–795
    [Google Scholar]
  21. Goldstein E. J., Agyare E. O., Vagvolgi A. E., Halpern M.. 1981; Aerobic bacterial oral flora of garter snakes: development of normal flora and pathogenic potential for snakes and humans. J Clin Microbiol13:954–956
    [Google Scholar]
  22. Hanahan D.. 1983; Studies on transformation of Escherichia coli with plasmids. J Mol Biol166:557–580
    [Google Scholar]
  23. Harrington A. T., Hearn P. D., Picking W. L., Barker J. R., Wessel A., Picking W. D.. 2003; Structural characterization of the N terminus of IpaC from Shigella flexneri . Infect Immun71:1255–1264
    [Google Scholar]
  24. Ide T., Laarmann S., Greune L., Schillers H., Oberleithner H., Schmidt M. A.. 2001; Characterization of translocation pores inserted into plasma membranes by type III-secreted Esp proteins of enteropathogenic Escherichia coli . Cell Microbiol3:669–679
    [Google Scholar]
  25. Jackson M. W., Day J. B., Plano G. V.. 1998; YscB of Yersinia pestis functions as a specific chaperone for YopN. J Bacteriol180:4912–4921
    [Google Scholar]
  26. Janda J. M., Abbott S. L.. 1993; Infections associated with the genus Edwardsiella : the role of Edwardsiella tarda in human disease. Clin Infect Dis17:742–748
    [Google Scholar]
  27. Jones S., Thornton J. M.. 1995; Protein-protein interactions: a review of primer dimer structures. Prog Biophys Mol Biol63:31–65
    [Google Scholar]
  28. Kalogeraki V. S., Winans S. C.. 1997; Suicide plasmids containing promoterless reporter genes can simultaneously disrupt and create fusions to target genes of diverse bacteria. Gene188:69–75
    [Google Scholar]
  29. Kourany M., Vasquez M. A., Saenz R.. 1977; Edwardsiellosis in man and animals in Panama: clinical and epidemiological characteristics. Am J Trop Med Hyg26:1183–1190
    [Google Scholar]
  30. Ling S. H., Wang X. H., Xie L., Lim T. M., Leung K. Y.. 2000; Use of green fluorescent protein (GFP) to study the invasion pathways of Edwardsiella tarda in in vivo and in vitro fish models. Microbiology146:7–19
    [Google Scholar]
  31. Macnab R. M.. 2003; How bacteria assemble flagella. Annu Rev Microbiol57:77–100
    [Google Scholar]
  32. Ménard R., Sansonetti P. J., Parsot C., Vasselon T.. 1994; Extracellular association and cytoplasmic partitioning of the IpaB and IpaC invasins of S. flexneri . Cell79:515–525
    [Google Scholar]
  33. Miller V. L., Mekalanos J. J.. 1988; A novel suicide vector and its use in construction of insertion mutations: osmoregulation of outer membrane proteins and virulence determinants in Vibrio cholerae requires toxR . J Bacteriol170:2575–2583
    [Google Scholar]
  34. Miyazaki S., Kuroda Y., Yokoyama S.. 2002; Characterization and prediction of linker sequences of multi-domain proteins by a neural network. J Struct Funct Genomics2:37–51
    [Google Scholar]
  35. Mo Z. L., Xiao P., Mao Y. X., Zou Y. X., Wang B., Li J., Xu Y. L., Zhang P. J.. 2007; Construction and characterization of a live, attenuated esrB mutant of Edwardsiella tarda and its potential as a vaccine against the haemorrhagic septicaemia in turbot, Scophthamus maximus (L.). Fish Shellfish Immunol23:521–530
    [Google Scholar]
  36. Neves B. C., Mundy R., Petrovska L., Dougan G., Knutton S., Frankel G.. 2003; CesD2 of enteropathogenic Escherichia coli is a second chaperone for the type III secretion translocator protein EspD. Infect Immun71:2130–2141
    [Google Scholar]
  37. Neyt C., Cornelis G. R.. 1999; Role of SycD, the chaperone of the Yersinia Yop translocators YopB and YopD. Mol Microbiol31:143–156
    [Google Scholar]
  38. Nikolaus T., Deiwichm J., Rappl C., Freeman J. A., Schröder W., Miller S. I., Hensel M.. 2001; SseBCD proteins are secreted by the type III secretion system of Salmonella pathogenicity island 2 and function as a translocon. J Bacteriol183:6036–6045
    [Google Scholar]
  39. Olsson J., Edqvist P. J., Bröms J. E., Forsberg A., Wolf-Watz H., Francis M. S.. 2004; The YopD translocator of Yersinia pseudotuberculosis is a multifunctional protein comprised of discrete domains. J Bacteriol186:4110–4123
    [Google Scholar]
  40. Page A. L., Parsot C.. 2002; Chaperones of the type III secretion pathway: jacks of all trades. Mol Microbiol46:1–11
    [Google Scholar]
  41. Pallen M. J., Beatson S. A., Bailey C. M.. 2005; Bioinformatics, genomics and evolution of non-flagellar type-III secretion systems: a Darwinian perspective. FEMS Microbiol Rev29:201–229
    [Google Scholar]
  42. Parsot C., Hamiaux C., Page A. L.. 2003; The various and varying roles of specific chaperones in type III secretion systems. Curr Opin Microbiol6:7–14
    [Google Scholar]
  43. Reed L. J., Muench H.. 1938; A simple method of estimating fifty percent end points. Am J Hyg27:493–497
    [Google Scholar]
  44. Rubirés X., Saigi F., Piqué N., Climent N., Merino S., Albertí S., Tomás J. M., Regué M.. 1997; A gene ( wbbL ) from Serratia marcescens N28b (O4) complements the rfb-50 mutation of Escherichia coli K-12 derivatives. J Bacteriol179:7581–7586
    [Google Scholar]
  45. Sae-Oui D., Muroga K., Nakai T.. 1984; A case of Edwardsiella tarda infection in cultured colored carp Cyprinus carpio . Fish Pathol19:197–199
    [Google Scholar]
  46. Saier M. H. Jr. 2004; Evolution of bacterial type III protein secretion systems. Trends Microbiol12:113–115
    [Google Scholar]
  47. Simon R., Priefer U., Pühler A.. 1983; A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in Gram-negative bacteria. Biotechnology1:784–791
    [Google Scholar]
  48. Srinivasa Rao P. S., Lim T. M., Leung K. Y.. 2001; Opsonized virulent Edwardsiella tarda strains are able to adhere to and survive and replicate within fish phagocytes but fail to stimulate reactive oxygen intermediates. Infect Immun69:5689–5697
    [Google Scholar]
  49. Srinivasa Rao P. S., Yamada Y., Leung K. Y.. 2003; A major catalase (KatB) that is required for resistance to H2O2 and phagocyte-mediated killing in Edwardsiella tarda . Microbiology149:2635–2644
    [Google Scholar]
  50. Srinivasa Rao P. S., Yamada Y., Tan Y. P., Leung K. Y.. 2004; Use of proteomics to identify novel virulence determinants that are required for Edwardsiella tarda pathogenesis. Mol Microbiol53:573–586
    [Google Scholar]
  51. Tan Y. P., Zheng J., Tung S. L., Rosenshine I., Leung K. Y.. 2005; Role of type III secretion in Edwardsiella tarda virulence. Microbiology151:2301–2313
    [Google Scholar]
  52. Thomas J., Stafford G. P., Hughes C.. 2004; Docking of cytosolic chaperone-substrate complexes at the membrane ATPase during flagellar type III protein export. Proc Natl Acad Sci U S A101:3945–3950
    [Google Scholar]
  53. Thomas N. A., Deng W., Puente J. L., Frey E. A., Yip C. K., Strynadka N. C., Finlay B. B.. 2005; CesT is a multi-effector chaperone and recruitment factor required for the efficient type III secretion of both LEE- and non-LEE-encoded effectors of enteropathogenic Escherichia coli . Mol Microbiol57:1762–1779
    [Google Scholar]
  54. Thune R. L., Stanley L. A., Cooper R. K.. 1993; Pathogenesis of gram-negative bacterial infections in warm water fish. Annu Rev Fish Dis3:37–68
    [Google Scholar]
  55. Towbin H., Staehelin T., Gordon J.. 1979; Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A76:4350–4354
    [Google Scholar]
  56. Tucker S. C., Galán J. E.. 2000; Complex function for SicA, a Salmonella enterica serovar Typhimurium type III secretion-associated chaperone. J Bacteriol182:2262–2268
    [Google Scholar]
  57. Wainwright L. A., Kaper J. B.. 1998; EspB and EspD require a specific chaperone for proper secretion from enteropathogenic E. coli . Mol Microbiol27:1247–1260
    [Google Scholar]
  58. Waterman S. R., Holden D. W.. 2003; Functions and effectors of the Salmonella pathogenicity island 2 type III secretion system. Cell Microbiol5:501–511
    [Google Scholar]
  59. Yang C. H., Wang C. K.. 1999; Edwardsiella tarda bacteraemia complicated by acute pancreatitis and pyomyoma. J Infect38:124–126
    [Google Scholar]
  60. Zheng J., Leung K. Y.. 2007; Dissection of a type VI secretion system in Edwardsiella tarda . Mol Microbiol66:1192–1206
    [Google Scholar]
  61. Zheng J., Li N., Tan Y. P., Sivaraman J., Mok Y. K., Mo Z. L., Leung K. Y.. 2007; EscC is a chaperone for the Edwardsiella tarda T3SS putative translocon components EseB and EseD. Microbiology153:1953–1962
    [Google Scholar]
  62. Zurawski D. V., Stein M. A.. 2003; SseA acts as the chaperone for the SseB component of the Salmonella pathogenicity island 2 translocon. Mol Microbiol47:1341–1351
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.021865-0
Loading
/content/journal/micro/10.1099/mic.0.021865-0
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