1887

Abstract

Enteropathogenic and enterohaemorrhagic (EPEC and EHEC, respectively) genomes contain a pathogenicity island, termed the locus of enterocyte effacement (LEE), which encodes genes involved in the formation of attaching and effacing lesions on epithelial cells. To elucidate the regulatory mechanism of the LEE genes in EHEC, an EHEC O157 genomic library was screened for clones which modulated expression of the LEE genes. From more than 5000 clones, a DNA fragment was obtained containing a homologue as a positive regulator for the LEE genes. In EPEC, is known to be part of the operon, along with and , located on the EPEC adherence factor plasmid, which is not found in EHEC. However, the complete genome sequence of EHEC O157 Sakai strain reveals that there are five -like sequences, but no and , on the chromosome. These five homologues were characterized, and it was found that three of the homologues (renamed omologue , and ) encoded 104 aa proteins, and when expressed on a multicopy plasmid enhanced the expression of LEE genes. In contrast, homologues encoding proteins of 89 and 90 aa, renamed and , respectively, had no significant effect. Deletion mutants of the genes were constructed, and the effect on the expression of LEE-encoded type III effector proteins, such as EspA, B and D, and adhesion phenotype to HEp-2 cells was examined. Deletion of or , but not , decreased the expression of Esp proteins and adhesion to HEp-2 cells. Such effects were more apparent with mutants carrying double deletions of / or /, suggesting that // are all necessary for full expression of the LEE genes and adhesion to HEp-2 cells. Further study demonstrated that the positive effect of // was caused by enhanced transcription of the LEE-encoded regulatory gene, . Introduction of a multicopy plasmid carrying each // gene significantly induced microcolony formation by EHEC O157 on HEp-2 cells. These results suggest that the genes are necessary for full virulence of EHEC O157.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.27100-0
2004-07-01
2019-10-18
Loading full text...

Full text loading...

/deliver/fulltext/micro/150/7/mic1502357.html?itemId=/content/journal/micro/10.1099/mic.0.27100-0&mimeType=html&fmt=ahah

References

  1. Bolivar, F., Rodriguez, R. L., Greene, P. J., Betlach, M. C., Heyneker, H. L., Boyer, H. W., Crosa, J. H. & Falkow, S. ( 1977; ). Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene 2, 95–113.[CrossRef]
    [Google Scholar]
  2. Bustamante, V., Santana, F., Calva, E. & Puente, J. L. ( 2001; ). Transcriptional regulation of type III secretion genes in enteropathogenic Escherichia coli: Ler antagonizes H-NS-dependent repression. Mol Microbiol 39, 664–678.[CrossRef]
    [Google Scholar]
  3. Chang, A. C. Y. & Cohen, S. N. ( 1978; ). Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. J Bacteriol 134, 1141–1156.
    [Google Scholar]
  4. Datsenko, K. A. & Wanner, B. I. ( 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.[CrossRef]
    [Google Scholar]
  5. Elliot, S. J., Wainwright, L. A., McDaniel, T., Macnamara, B., Konnenberg, M. & 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]
  6. Elliott, S. J., Hutcheson, S. W., Dubois, M. S., Mellies, J. L., Wainwright, L. A., Batchelor, M., Frankel, G., Knutton, S. & Kaper, J. B. ( 1999a; ). Identification of CesT, a chaperone for the type III secretion of Tir in enteropathogenic Escherichia coli. Mol Microbiol 33, 1176–1188.
    [Google Scholar]
  7. Elliott, S. J., Yu, J. & Kaper, J. B. ( 1999b; ). The cloned locus of enterocyte effacement (LEE) from enterohemorrhagic Escherichia coli O157 : H7 is unable to confer the attaching and effacing phenotype upon E. coli K-12. Infect Immun 67, 4260–4263.
    [Google Scholar]
  8. Elliott, S. J., Sperandio, V., Girón, J. A., Shin, S., Mellies, J. L., Wainwright, L. A., Hutcheson, S. W., McDaniel, T. K. & Kaper, J. B. ( 2000; ). The locus of enterocyte effacement (LEE)-encoded regulator (Ler) controls expression of both LEE- and non-LEE-encoded virulence factors in enteropathogenic and enterohemorrhagic Escherichia coli. Infect Immun 68, 6115–6126.[CrossRef]
    [Google Scholar]
  9. Favre, D. & Viret, J. ( 1996; ). Versatile cosmid vectors for facilitated analysis and subcloning of the insert. Gene 178, 43–49.[CrossRef]
    [Google Scholar]
  10. 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.[CrossRef]
    [Google Scholar]
  11. Friedberg, D., Umanski, T., Fang, Y. & Rosenshine, I. ( 1999; ). Hierarchy in the expression of the locus of enterocyte effacement genes of enteropathogenic Escherichia coli. Mol Microbiol 34, 941–952.[CrossRef]
    [Google Scholar]
  12. Goldberg, M. D., Johnson, M., Hinton, J. C. & Williams, P. H. ( 2001; ). Role of the nucleoid-associated protein Fis in the regulation of virulence properties of enteropathogenic Escherichia coli. Mol Microbiol 41, 549–559.[CrossRef]
    [Google Scholar]
  13. Gómez-Duarte, O. G. & Kaper, J. B. ( 1995; ). A plasmid-encoded regulatory region activates chromosomal eaeA expression in enteropathogenic Escherichia coli. Infect Immun 63, 1767–1776.
    [Google Scholar]
  14. Haak, K. R., Robinson, C. L., Miller, K. J., Fowlkes, J. W. & Mellies, J. L. ( 2003; ). Interaction of Ler at the LEE5 (tir) operon of enteropathogenic Escherichia coli. Infect Immun 71, 384–392.[CrossRef]
    [Google Scholar]
  15. Hayashi, T., Makino, K., Ohnishi, M. & 19 other authors ( 2001a; ). Complete genome sequence of enterohemorrhagic Escherichia coli O157 : H7 and genomic comparison with a laboratory strain K-12. DNA Res 8, 11–22.[CrossRef]
    [Google Scholar]
  16. Hayashi, T., Makino, K., Ohnishi, M. & 19 other authors ( 2001b; ). Complete genome sequence of enterohemorrhagic Escherichia coli O157 : H7 and genomic comparison with a laboratory strain K-12 (Supplement). DNA Res 8, 47–52.[CrossRef]
    [Google Scholar]
  17. Iyoda, S. & Kutsukake, K. ( 1995; ). Molecular dissection of the flagellum-specific anti-sigma factor, FlgM, of Salmonella typhimurium. Mol Gen Genet 249, 417–424.
    [Google Scholar]
  18. Iyoda, S., Kamidoi, T., Hirose, K., Kutsukake, K. & Watanabe, H. ( 2001; ). A flagellar gene fliZ regulates the expression of invasion genes and virulence phenotype in Salmonella enterica serovar Typhimurium. Microb Pathog 30, 81–90.[CrossRef]
    [Google Scholar]
  19. Jarvis, K. G., Girón, J. A., Jerse, A. E., McDaniel, T. K., Donnenberg, M. S. & Kaper, J. B. ( 1995; ). Enteropathogenic Escherichia coli contains a specialized secretion system necessary for the export of proteins involved in attaching and effacing lesion formation. Proc Natl Acad Sci U S A 92, 7996–8000.[CrossRef]
    [Google Scholar]
  20. Jerse, A. E. & Kaper, J. B. ( 1991; ). The eae gene of enteropathogenic Escherichia coli encodes a 94-kilodalton membrane protein, the expression of which is influenced by the EAF plasmid. Infect Immun 59, 4302–4309.
    [Google Scholar]
  21. 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.[CrossRef]
    [Google Scholar]
  22. Kanamaru, K., Kanamaru, K., Tatsuno, I., Tobe, T. & Sasakawa, C. ( 2000; ). SdiA, an Escherichia coli homologue of quorum-sensing regulators, controls the expression of virulence factors in enterohemorrhagic Escherichia coli O157 : H7. Mol Microbiol 38, 805–816.[CrossRef]
    [Google Scholar]
  23. Kenny, B. & Finlay, B. B. ( 1995; ). Protein secretion by enteropathogenic Escherichia coli is essential for transducing signals to epithelial cells. Proc Natl Acad Sci U S A 92, 7991–7995.[CrossRef]
    [Google Scholar]
  24. Kenny, B., Lai, L. C., Finlay, B. B. & Donnenberg, M. S. ( 1996; ). EspA, a protein secreted by enteropathogenic Escherichia coli, is required to induce signals in epithelial cells. Mol Microbiol 20, 313–323.[CrossRef]
    [Google Scholar]
  25. Kenny, B., DeVinney, R., Stein, M., Reinscheid, D. J., Frey, E. A. & Finlay, B. B. ( 1997; ). Enteropathogenic E. coli (EPEC) transfers its receptor for intimate adherence into mammalian cells. Cell 91, 511–520.[CrossRef]
    [Google Scholar]
  26. Knutton, S., Lloyd, D. R. & McNeish, A. S. ( 1987; ). Adhesion of enteropathogenic Escherichia coli to human intestinal enterocytes and cultured human intestinal mucosa. Infect Immun 55, 69–77.
    [Google Scholar]
  27. Koop, A. H., Hartley, M. E. & Bourgeois, S. ( 1987; ). A low-copy-number vector utilizing β-galactosidase for the analysis of gene control elements. Gene 52, 245–256.[CrossRef]
    [Google Scholar]
  28. Lai, L. C., Wainwright, L. A., Stone, K. D. & Donnenberg, M. S. ( 1997; ). A third secreted protein that is encoded by the enteropathogenic Escherichia coli pathogenicity island is required for transduction of signals and for attaching and effacing activities in host cells. Infect Immun 65, 2211–2217.
    [Google Scholar]
  29. Martinez-Laguna, Y., Calva, E. & Puente, J. L. ( 1999; ). Autoactivation and environmental regulation of bfpT expression, the gene coding for the transcriptional activator of bfpA in enteropathogenic Escherichia coli. Mol Microbiol 33, 153–166.[CrossRef]
    [Google Scholar]
  30. McDaniel, T. K. & Kaper, J. B. ( 1997; ). A cloned pathogenicity island from enteropathogenic Escherichia coli confers the attaching and effacing phenotype on E. coli K-12. Mol Microbiol 23, 399–407.[CrossRef]
    [Google Scholar]
  31. 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.[CrossRef]
    [Google Scholar]
  32. McNamara, B. P. & Donnenberg, M. S. ( 1998; ). A novel proline-rich protein, EspF, is secreted from enteropathogenic Escherichia coli via the type III export pathway. FEMS Microbiol Lett 166, 71–78.[CrossRef]
    [Google Scholar]
  33. 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.[CrossRef]
    [Google Scholar]
  34. Moon, H. W., Whipp, S. C., Argenzio, R. A., Levine, M. M. & Giannella, R. A. ( 1983; ). Attaching and effacing activities of rabbit and human enteropathogenic Escherichia coli in pig and rabbit intestines. Infect Immun 41, 1340–1351.
    [Google Scholar]
  35. Nataro, J. P. & Kaper, J. B. ( 1998; ). Diarrheagenic Escherichia coli. Clin Microbiol Rev 11, 142–201.
    [Google Scholar]
  36. Ohnishi, M., Kurokawa, K. & Hayashi, T. ( 2001; ). Diversification of Escherichia coli genomes: are bacteriophages the major contributors? Trends Microbiol 9, 481–485.[CrossRef]
    [Google Scholar]
  37. Perna, N. T., Plunkett, G., Burland, V. & 25 other authors ( 2001; ). Genome sequence of enterohaemorrhagic Escherichia coli O157 : H7. Nature 409, 529–533.[CrossRef]
    [Google Scholar]
  38. Reece, K. S. & Phillips, G. J. ( 1995; ). New plasmids carrying antibiotic-resistance cassettes. Gene 165, 141–142.[CrossRef]
    [Google Scholar]
  39. Rosenshine, I., Ruschkowski, S., Stein, M., Reinscheid, D. J., Mills, S. D. & Finlay, B. B. ( 1996; ). A pathogenic bacterium triggers epithelial signals to form a functional bacterial receptor that mediates actin pseudopod formation. EMBO J 15, 2613–2624.
    [Google Scholar]
  40. Sanchez-SanMartin, C., Bustamante, V. H., Calva, E. & Puente, J. L. ( 2001; ). Transcriptional regulation of the orf19 gene and the tir-cesT-eae operon of enteropathogenic Escherichia coli. J Bacteriol 183, 2823–2833.[CrossRef]
    [Google Scholar]
  41. Shin, S., Castanie-Cornet, M. P., Foster, J. W., Crawford, J. A., Brinkley, C. & Kaper, J. B. ( 2001; ). An activator of glutamate decarboxylase genes regulates the expression of enteropathogenic Escherichia coli virulence genes through control of the plasmid-encoded regulator, Per. Mol Microbiol 41, 1133–1150.
    [Google Scholar]
  42. Sohel, I., Puente, J. L., Ramer, S. W., Bieber, D., Wu, C.-Y. & Schoolnik, G. K. ( 1996; ). Enteropathogenic Escherichia coli: identification of a gene cluster coding for bundle-forming pilus morphogenesis. J Bacteriol 178, 2613–2628.
    [Google Scholar]
  43. Sperandio, V., Mellies, J. L., Nguyen, W., Shin, S. & Kaper, J. B. ( 1999; ). Quorum sensing controls expression of the type III secretion gene transcription and protein secretion in enterohemorrhagic and enteropathogenic Escherichia coli. Proc Natl Acad Sci U S A 96, 15196–15201.[CrossRef]
    [Google Scholar]
  44. Sperandio, V., Mellies, J. L., Delahay, R. M., Frankel, G., Crawford, J. A., Nguyen, W. & Kape, J. B. ( 2000; ). Activation of enteropathogenic Escherichia coli (EPEC) LEE2 and LEE3 operons by Ler. Mol Microbiol 38, 781–793.[CrossRef]
    [Google Scholar]
  45. Sperandio, V., Li, C. C. & Kaper, J. B. ( 2002; ). Quorum-sensing Escherichia coli regulator A: a regulator of the LysR family involved in the regulation of the locus of enterocyte effacement pathogenicity island in enterohemorrhagic E. coli. Infect Immun 70, 3085–3093.[CrossRef]
    [Google Scholar]
  46. Stachel, S. E., An, G., Flores, C. & Nester, E. W. ( 1985; ). A Tn3 lacZ transposon for the random generation of beta-galactosidase gene fusions: application to the analysis of gene expression in Agrobacterium. EMBO J 4, 891–898.
    [Google Scholar]
  47. Stone, K. D., Zhang, H. Z., Carlson, L. K. & Donnenberg, M. S. ( 1996; ). A cluster of fourteen genes from enteropathogenic Escherichia coli is sufficient for the biogenesis of a type IV pilus. Mol Microbiol 20, 325–337.[CrossRef]
    [Google Scholar]
  48. Tatsuno, I., Nagano, K., Taguchi, K., Rong, L., Mori, H. & Sasakawa, C. ( 2003; ). Increased adherence to Caco-2 cells caused by disruption of the yhiE and yhiF genes in enterohemorrhagic Escherichia coli O157 : H7. Infect Immun 71, 2598–2606.[CrossRef]
    [Google Scholar]
  49. Tobe, T., Schoolnik, G. K., Sohel, I., Bustamante, V. H. & Puente, J. L. ( 1996; ). Cloning and characterization of bfpTVW, genes required for the transcriptional activation of bfpA in enteropathogenic Escherichia coli. Mol Microbiol 21, 963–975.[CrossRef]
    [Google Scholar]
  50. Tzipori, S., Wachsmuth, I. K., Chapman, C., Birden, R., Brittingham, J., Jackson, C. & Hogg, J. ( 1986; ). The pathogenesis of hemorrhagic colitis caused by Escherichia coli O157 : H7 in gnotobiotic piglets. J Infect Dis 154, 712–714.[CrossRef]
    [Google Scholar]
  51. Umanski, T., Rosenshine, I. & Friedberg, D. ( 2002; ). Thermoregulated expression of virulence genes in enteropathogenic Escherichia coli. Microbiology 148, 2735–2744.
    [Google Scholar]
  52. Yanisch-Perron, C., Vieira, J. & Messing, J. ( 1985; ). Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33, 103–119.[CrossRef]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.27100-0
Loading
/content/journal/micro/10.1099/mic.0.27100-0
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