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Volume 149, Issue 8

Yeast two-hybrid system survey of interactions between LEE-encoded proteins of enteropathogenic Free

Abstract

Many Gram-negative pathogens employ a specific secretion pathway, termed type III secretion, to deliver virulence effector proteins directly to the membranes and cytosol of host eukaryotic cells. Subsequent functions of many effector proteins delivered in this manner result in subversion of host-signalling pathways to facilitate bacterial entry, survival and dissemination to neighbouring cells and tissues. Whereas the secreted components of type III secretion systems (TTSSs) from different pathogens are structurally and functionally diverse, the structural components and the secretion apparatus itself are largely conserved. TTSSs are large macromolecular assemblies built through interactions between protein components of hundreds of individual subunits. The goal of this project was to screen, using the standard yeast two-hybrid system, pair-wise interactions between components of the enteropathogenic TTSS. To this end 37 of the 41 genes encoded by the LEE pathogenicity island were cloned into both yeast two-hybrid system vectors and all possible permutations of interacting protein pairs were screened for. This paper reports the identification of 22 novel interactions, including interactions between inner-membrane structural TTSS proteins; between the type III secreted translocator protein EspD and structural TTSS proteins; between established and putative chaperones and their cognate secreted proteins; and between proteins of undefined function.

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Keyword(s): A/E, attaching and effacing , EPEC, enteropathogenic Escherichia coli , LEE, locus of enterocyte effacement , NC, needle complex , TTSS, type III secretion system and Y2HS, yeast two-hybrid system
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2003-08-01
2024-04-19
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References

  1. Abe A., de Grado M., Pfuetzner R. A., Sanchez-SanMartin C., DeVinney R., Puente J. L., Strynadka N. C. J., Finlay B. B. 1999; Enteropathogenic Escherichia coli translocated intimin receptor, Tir, requires a specific chaperone for stable secretion. Mol Microbiol 33:1162–1175
     [Google Scholar]
  2. 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
     [Google Scholar]
  3. Auvray F., Thomas J., Fraser G. M., Hughes C. 2001; Flagellin polymerisation control by a cytosolic export chaperone. J Mol Biol 308:221–229
     [Google Scholar]
  4. 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
     [Google Scholar]
  5. Brent R., Finley R. L. 1997; Understanding gene and allele function with two-hybrid methods. Annu Rev Genet 31:663–704
     [Google Scholar]
  6. Buttner D., Bonas U. 2002; Port of entry – the type III secretion translocon. Trends Microbiol 10:186–192
     [Google Scholar]
  7. Claros M. G., von Heijne G. 1994; TopPred II: an improved software for membrane protein structure predictions. Comput Appl Biosci 10:685–686
     [Google Scholar]
  8. Crago A. M., Koronakis V. 1998; Salmonella InvG forms a ring-like multimer that requires the InvH lipoprotein for outer membrane localization. Mol Microbiol 30:47–56
     [Google Scholar]
  9. Creasey E. A., Delahay R. M., Bishop A. A., Shaw R. K., Kenny B., Knutton S., Frankel G. 2003; CesT is a bivalent enteropathogenic Escherichia coli chaperone required for translocation of both Tir and Map. Mol Microbiol 47:209–221
     [Google Scholar]
  10. Cserzo M., Wallin E., Simon I., von Heijne G., Elofsson A. 1997; Prediction of transmembrane alpha-helices in prokaryotic membrane proteins: the dense alignment surface method. Protein Eng 10:673–676
     [Google Scholar]
  11. 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 Immun 69:4055–4064
     [Google Scholar]
  12. De Grado M., Abe A., Gauthier A., Steele-Mortimer O., DeVinney R., Finlay B. B. 1999; Identification of the intimin-binding domain of Tir of enteropathogenic Escherichia coli . Cell Microbiol 1:7–17
     [Google Scholar]
  13. Delahay R. M., Shaw R. K., Elliott S. J., Kaper J. B., Knutton S., Frankel G. 2002; Functional analysis of the enteropathogenic Escherichia coli type III secretion system chaperone CesT identifies domains that mediate substrate interactions. Mol Microbiol 43:61–73
     [Google Scholar]
  14. Drees B. L. 1999; Progress and variations in two-hybrid and three-hybrid technologies. Curr Opin Chem Biol 3:64–70
     [Google Scholar]
  15. Elliott S. J., Wainwright L. A., McDaniel T. K., Jarvis K. G., Deng Y., Lai L. C., McNamara B. P., Donnenberg M. S., Kaper J. B. 1998; The complete sequence of the locus of enterocyte effacement (LEE) from enteropathogenic Escherichia coli E2348/69. Mol Microbiol 28:1–4
     [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 Microbiol 33:1176–1189
     [Google Scholar]
  17. Elliott S. J., O'Connell C. B., Koutsouris A., Brinkley C., Donnenberg M. S., Hecht G., Kaper J. B. 2002; A gene from the locus of enterocyte effacement that is required for enteropathogenic Escherichia coli to increase tight-junction permeability encodes a chaperone for EspF. Infect Immun 70:2271–2277
     [Google Scholar]
  18. Francis M. S., Aili M., Wiklund M. L., Wolf-Watz H. 2000; A study of the YopD-LcrH interaction from Yersinia pseudotuberculosis reveals a role for hydrophobic residues within the amphipathic domain of YopD. Mol Microbiol 38:85–102
     [Google Scholar]
  19. Francis M. S., Lloyd S. A., Wolf-Watz H. 2001; The type III secretion chaperone LcrH co-operates with YopD to establish a negative, regulatory loop for control of Yop synthesis in Yersinia pseudotuberculosis . Mol Microbiol 42:1075–1093
     [Google Scholar]
  20. Frankel G., Phillips A. D., Rosenshine I., Dougan G., Kaper J. B., Knutton S. 1998; Enteropathogenic and enterohaemorrhagic Escherichia coli : more subversive elements. Mol Microbiol 30:911–921
     [Google Scholar]
  21. Genin S., Boucher C. A. 1994; A superfamily of proteins involved in different secretion pathways in gram-negative bacteria: modular structure and specificity of the N-terminal domain. Mol Gen Genet 243:112–118
     [Google Scholar]
  22. Gietz R. D., Schiestl R. H. 1995; Transforming yeast with DNA. Methods Mol Cell Biol 5:255–269
     [Google Scholar]
  23. Hartland E. L., Batchelor M., Delahay R. M., Hale C., Matthews S., Dougan G., Knutton S., Connerton I., Frankel G. 1999; Binding of intimin from enteropathogenic Escherichia coli to Tir and to host cells. Mol Microbiol 32:151–158
     [Google Scholar]
  24. Hartland E. L., Daniell S. J., Delahay R. M., Neves B. C., Wallis T., Shaw R. K., Hale C., Knutton S., Frankel G. 2000; The type III protein translocation system of enteropathogenic Escherichia coli involves EspA-EspB protein interactions. Mol Microbiol 35:1483–1492
     [Google Scholar]
  25. Hueck C. J. 1998; Type III protein secretion systems in bacterial pathogens of animals and plants. Microbiol Mol Biol Rev 62:379–433
     [Google Scholar]
  26. 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 Microbiol 3:669–679
     [Google Scholar]
  27. Jackson M. W., Plano G. V. 2000; Interactions between type III secretion apparatus components from Yersinia pestis detected using the yeast two-hybrid system. FEMS Microbiol Lett 186:85–90
     [Google Scholar]
  28. James P., Halladay J., Craig E. A. 1996; Genomic libraries and a host strain designed for highly efficient two-hybrid selection in yeast. Genetics 144:1425–1436
     [Google Scholar]
  29. Jerse A. E., Yu J., Tall B. D., Kaper J. B. 1990; A genetic locus of enteropathogenic Escherichia coli necessary for the production of attaching and effacing lesions on tissue culture cells. Proc Natl Acad Sci U S A 87:7839–7843
     [Google Scholar]
  30. Kenny B., Jepson M. 2000; Targeting of an enteropathogenic Escherichia coli (EPEC) effector protein to host mitochondria. Cell Microbiol 2:579–590
     [Google Scholar]
  31. Knutton S., Rosenshine I., Pallen M. J., Nisan I., Neves B. C., Bain C., Wolff C., Dougan G., Frankel G. 1998; A novel EspA-associated surface organelle of enteropathogenic Escherichia coli involved in protein translocation into epithelial cells. EMBO J 17:2166–2176
     [Google Scholar]
  32. Kodama T., Akeda Y., Kono G., Takahashi A., Imura K., Iida T., Honda T. 2002; The EspB protein of enterohaemorrhagic Escherichia coli interacts directly with α -catenin. Cell Microbiol 4:213–222
     [Google Scholar]
  33. Koster M., Bitter W., de Cock H., Allaoui A., Cornelis G. R., Tommassen J. 1997; The outer membrane component, YscC, of the Yop secretion machinery of Yersinia enterocolitica forms a ring-shaped multimeric complex. Mol Microbiol 26:789–797
     [Google Scholar]
  34. Kresse A. U., Rohde M., Guzman C. A. 1999; The EspD protein of enterohemorrhagic Escherichia coli is required for the formation of bacterial surface appendages and is incorporated in the cytoplasmic membranes of target cells. Infect Immun 67:4834–4842
     [Google Scholar]
  35. Krogh A., Larsson B., von Heijne G., Sonnhammer E. L. 2001; Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes. J Mol Biol 305:567–580
     [Google Scholar]
  36. Levine M. M., Bergquist E. J., Nalin D. R., Waterman D. H., Hornick R. B., Young C. R., Sotman S. 1978; Escherichia coli strains that cause diarrhoea but do not produce heat-labile or heat-stable enterotoxins and are non-invasive. Lancet 1:1119–1122
     [Google Scholar]
  37. Luo Y., Frey E. A., Pfuetzner R. A., Creagh A. L., Knoechel D. G., Haynes C. A., Finlay B. B., Strynadka N. C. J. 2000; Crystal structure of enteropathogenic Escherichia coli intimin-receptor complex. Nature 405:1073–1077
     [Google Scholar]
  38. McDaniel T. K., Jarvis K. G., Donnenberg M. S., Kaper J. B. 1995; A genetic locus of enterocyte effacement conserved among diverse enterobacterial pathogens. Proc Natl Acad Sci U S A 92:1664–1668
     [Google Scholar]
  39. Mellies J. L., Elliott S. J., Sperandio V., Donnenberg M. S., Kaper J. B. 1999; The Per regulon of enteropathogenic Escherichia coli : identification of a regulatory cascade and a novel transcriptional activator, the locus of enterocyte effacement (LEE)-encoded regulator (Ler. Mol Microbiol 33:296–306
     [Google Scholar]
  40. Miller J. H. 1972 Experiments in Molecular Genetics Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
  41. Minamino T., Macnab R. M. 1999; Components of the Salmonella flagellar export apparatus and classification of export substrates. J Bacteriol 181:1388–1394
     [Google Scholar]
  42. Minamino T., Macnab R. M. 2000a; Interactions among components of the Salmonella flagellar export apparatus and its substrates. Mol Microbiol 35:1052–1064
     [Google Scholar]
  43. Minamino T., Macnab R. M. 2000b; Domain structure of Salmonella FlhB, a flagellar export component responsible for substrate specificity switching. J Bacteriol 182:4906–4914
     [Google Scholar]
  44. 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 Immun 71:2130–2141
     [Google Scholar]
  45. Neyt C., Cornelis G. R. 1999; Role of SycD, the chaperone of the Yersinia Yop translocators YopB and YopD. Mol Microbiol 31:143–156
     [Google Scholar]
  46. Nielsen H., Engelbrecht J., Brunak S., von Heijne G. 1997; A neural network method for identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Int J Neural Syst 8:581–599
     [Google Scholar]
  47. Nouwen N., Ranson N., Saibil H., Wolpensinger B., Engel A., Ghazi A., Pugsley A. P. 1999; Secretin PulD: association with pilot PulS, structure and ion-conducting channel formation. Proc Natl Acad Sci U S A 96:8173–8177
     [Google Scholar]
  48. Nouwen N., Stahlberg H., Pugsley A. P., Engel A. 2000; Domain structure of secretin PulD revealed by limited proteolysis and electron microscopy. EMBO J 19:2229–2236
     [Google Scholar]
  49. 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 Microbiol 42:1133–1145
     [Google Scholar]
  50. Page A. L., Sansonetti P., Parsot C. 2002; Spa15 of Shigella flexneri , a third type of chaperone in the type III secretion pathway. Mol Microbiol 43:1533–1542
     [Google Scholar]
  51. Schuch R., Maurelli A. T. 2001; MxiM and MxiJ, Base elements of the Mxi-Spa type III secretion system of Shigella , interact with and stabilize the MxiD secretin in the cell envelope. J Bacteriol 183:6991–6998
     [Google Scholar]
  52. Sekiya K., Ohishi M., Ogino T., Tamano K., Sasakawa C., Abe A. 2001; Supermolecular structure of the enteropathogenic Escherichia coli type III secretion system and its direct interaction with the EspA-sheath-like structure. Proc Natl Acad Sci U S A 98:11638–11643
     [Google Scholar]
  53. Sonnhammer E. L., von Heijne G., Krogh A. 1998; A hidden Markov model for predicting transmembrane helices in protein sequences. Proc Int Conf Intell Syst Mol Biol 6:175–182
     [Google Scholar]
  54. Sukhan A., Kubori T., Wilson J., Galan J. E. 2001; Genetic analysis of assembly of the Salmonella enterica serovar Typhimurium type III secretion-associated needle complex. J Bacteriol 183:1159–1167
     [Google Scholar]
  55. Suzuki H., Yonekura K., Murata K., Hirai T., Oosawa K., Namba K. 1998; A structural feature in the central channel of the bacterial flagellar FliF ring complex is implicated in type III protein export. J Struct Biol 124:104–114
     [Google Scholar]
  56. Thanassi D. G., Hultgren S. J. 2000; Multiple pathways allow protein secretion across the bacterial outer membrane. Curr Opin Cell Biol 12:420–430
     [Google Scholar]
  57. Tucker S. C., Galan J. E. 2000; Complex function for SicA, a Salmonella enterica serovar Typhimurium type III secretion-associated chaperone. J Bacteriol 182:2262–2268
     [Google Scholar]
  58. Tusnady G. E., Simon I. 2001; The HMMTOP transmembrane topology prediction server. Bioinformatics 17:849–850
     [Google Scholar]
  59. Wainwright L. A., Kaper J. B. 1998; EspB and EspD require a specific chaperone for proper secretion from enteropathogenic Escherichia coli . Mol Microbiol 27:1247–1260
     [Google Scholar]
  60. Wilkins M. R., Gasteiger E., Wheeler C. H., Lindskog I., Sanchez J. C., Bairoch A., Appel R. D., Dunn M. J., Hochstrasser D. F. 1998; Multiple parameter cross-species protein identification using MultiIdent – a world-wide web accessible tool. Electrophoresis 19:3199–3206
     [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 Bacteriol 178:2960–2970
     [Google Scholar]
  62. Wilson R. K., Shaw R. K., Daniell S., Knutton S., Frankel G. 2001; Role of EscF, a putative needle complex protein, in the type III protein translocation system of enteropathogenic Escherichia coli . Cell Microbiol 3:753–762
     [Google Scholar]
  63. Zhu C., Agin T. S., Elliott S. J., Johnson L. A., Thate T. E., Kaper J. B., Boedeker E. C. 2001; Complete nucleotide sequence and analysis of the locus of enterocyte effacement from rabbit diarrheagenic Escherichia coli RDEC-1. Infect Immun 69:2107–2115
     [Google Scholar]
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Yeast two-hybrid system survey of interactions between LEE-encoded proteins of enteropathogenic Escherichia coli
Microbiology 149, 2093 (2003); https://doi.org/10.1099/mic.0.26355-0
/content/journal/micro/10.1099/mic.0.26355-0
/content/journal/micro/10.1099/mic.0.26355-0
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