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. 1996 Nov;64(11):4795–4801. doi: 10.1128/iai.64.11.4795-4801.1996

Role of the Escherichia coli O157:H7 O side chain in adherence and analysis of an rfb locus.

S S Bilge 1, J C Vary Jr 1, S F Dowell 1, P I Tarr 1
PMCID: PMC174447  PMID: 8890241

Abstract

Shiga-toxigenic Escherichia coli strains belonging to serotype O157 are important human pathogens, but the genetic basis of expression of the O157 antigen and the role played by the lipopolysaccharide O side chain in the adherence of this organism to epithelial cells are not understood. We performed TnphoA mutagenesis on E. coli O157:H7 strain 86-24 to identify a mutant (strain F12) deficient in O-antigen expression. Nucleotide sequence analysis demonstrated that the transposon inserted within an open reading frame with significant homology to rfbE of Vibrio cholerae O1 (U. H. Stroeher, L. E. Karageorgos, R. Morona, and P. A. Manning, Proc. Natl. Acad. Sci. USA 89:2566-2570, 1992), which is postulated to encode perosamine synthetase. This open reading frame was designated rfbE(EcO157:H7). The guanine-plus-cytosine fraction (0.35) suggests that rfbE(EcO157:H7) may have originated in a species other than E. coli. rfbE(EcO157:H7) is conserved in nontoxigenic E. coli O157 strains expressing a variety of other flagellar antigens but is not found in E. coli O55:H7 strains, which are more closely related to E. coli O157:H7. Strain F12 was significantly more adherent to HeLa cells in a quantitative adherence assay than was its E. coli O157:H7 parent, but they did not differ in other phenotypes. Restoration of the expression of the O side chain by complementation of the TnphoA mutation in strain F12 by a plasmid expressing intact rfbE(EcO157:H7) reduced the adherence of the hyperadherent strain F12. We conclude that rfbE(EcO157:H7) is necessary for the expression of the O157 antigen, that acquisition of E. coli rfb genes occurred independently in E. coli O157:H7 and unrelated O157 strains, and that the O side chain of E. coli O157:H7 lipopolysaccharide interferes with the adherence of E. coli O157:H7 to epithelial cells.

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Selected References

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  1. Achtman M., Mercer A., Kusecek B., Pohl A., Heuzenroeder M., Aaronson W., Sutton A., Silver R. P. Six widespread bacterial clones among Escherichia coli K1 isolates. Infect Immun. 1983 Jan;39(1):315–335. doi: 10.1128/iai.39.1.315-335.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bachmann B. J. Linkage map of Escherichia coli K-12, edition 8. Microbiol Rev. 1990 Jun;54(2):130–197. doi: 10.1128/mr.54.2.130-197.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bell B. P., Goldoft M., Griffin P. M., Davis M. A., Gordon D. C., Tarr P. I., Bartleson C. A., Lewis J. H., Barrett T. J., Wells J. G. A multistate outbreak of Escherichia coli O157:H7-associated bloody diarrhea and hemolytic uremic syndrome from hamburgers. The Washington experience. JAMA. 1994 Nov 2;272(17):1349–1353. [PubMed] [Google Scholar]
  4. Bentley A. T., Klebba P. E. Effect of lipopolysaccharide structure on reactivity of antiporin monoclonal antibodies with the bacterial cell surface. J Bacteriol. 1988 Mar;170(3):1063–1068. doi: 10.1128/jb.170.3.1063-1068.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bisercić M., Feutrier J. Y., Reeves P. R. Nucleotide sequences of the gnd genes from nine natural isolates of Escherichia coli: evidence of intragenic recombination as a contributing factor in the evolution of the polymorphic gnd locus. J Bacteriol. 1991 Jun;173(12):3894–3900. doi: 10.1128/jb.173.12.3894-3900.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Blum G., Ott M., Lischewski A., Ritter A., Imrich H., Tschäpe H., Hacker J. Excision of large DNA regions termed pathogenicity islands from tRNA-specific loci in the chromosome of an Escherichia coli wild-type pathogen. Infect Immun. 1994 Feb;62(2):606–614. doi: 10.1128/iai.62.2.606-614.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Bokete T. N., O'Callahan C. M., Clausen C. R., Tang N. M., Tran N., Moseley S. L., Fritsche T. R., Tarr P. I. Shiga-like toxin-producing Escherichia coli in Seattle children: a prospective study. Gastroenterology. 1993 Dec;105(6):1724–1731. doi: 10.1016/0016-5085(93)91069-t. [DOI] [PubMed] [Google Scholar]
  8. Chart H., Rowe B. Antibody cross-reactions with lipopolysaccharide from E. coli O157 after cholera vaccination. Lancet. 1993 May 15;341(8855):1282–1282. doi: 10.1016/0140-6736(93)91187-q. [DOI] [PubMed] [Google Scholar]
  9. Cheah K. C., Manning P. A. Inactivation of the Escherichia coli B41 (O101:K99/F41) rfb gene encoding an 80-kDa polypeptide results in the synthesis of an antigenically altered lipopolysaccharide in E. coli K-12. Gene. 1993 Jan 15;123(1):9–15. doi: 10.1016/0378-1119(93)90532-8. [DOI] [PubMed] [Google Scholar]
  10. Cockerill F., 3rd, Beebakhee G., Soni R., Sherman P. Polysaccharide side chains are not required for attaching and effacing adhesion of Escherichia coli O157:H7. Infect Immun. 1996 Aug;64(8):3196–3200. doi: 10.1128/iai.64.8.3196-3200.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Devereux J., Haeberli P., Smithies O. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1):387–395. doi: 10.1093/nar/12.1part1.387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Gentry M. K., Dalrymple J. M. Quantitative microtiter cytotoxicity assay for Shigella toxin. J Clin Microbiol. 1980 Sep;12(3):361–366. doi: 10.1128/jcm.12.3.361-366.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Gish W., States D. J. Identification of protein coding regions by database similarity search. Nat Genet. 1993 Mar;3(3):266–272. doi: 10.1038/ng0393-266. [DOI] [PubMed] [Google Scholar]
  14. Gribskov M., Burgess R. R. Sigma factors from E. coli, B. subtilis, phage SP01, and phage T4 are homologous proteins. Nucleic Acids Res. 1986 Aug 26;14(16):6745–6763. doi: 10.1093/nar/14.16.6745. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Griffin P. M., Ostroff S. M., Tauxe R. V., Greene K. D., Wells J. G., Lewis J. H., Blake P. A. Illnesses associated with Escherichia coli O157:H7 infections. A broad clinical spectrum. Ann Intern Med. 1988 Nov 1;109(9):705–712. doi: 10.7326/0003-4819-109-9-705. [DOI] [PubMed] [Google Scholar]
  16. Gunzer F., Böhm H., Rüssmann H., Bitzan M., Aleksic S., Karch H. Molecular detection of sorbitol-fermenting Escherichia coli O157 in patients with hemolytic-uremic syndrome. J Clin Microbiol. 1992 Jul;30(7):1807–1810. doi: 10.1128/jcm.30.7.1807-1810.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Inzana T. J. Electrophoretic heterogeneity and interstrain variation of the lipopolysaccharide of Haemophilus influenzae. J Infect Dis. 1983 Sep;148(3):492–499. doi: 10.1093/infdis/148.3.492. [DOI] [PubMed] [Google Scholar]
  18. Kessler A. C., Haase A., Reeves P. R. Molecular analysis of the 3,6-dideoxyhexose pathway genes of Yersinia pseudotuberculosis serogroup IIA. J Bacteriol. 1993 Mar;175(5):1412–1422. doi: 10.1128/jb.175.5.1412-1422.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Marolda C. L., Valvano M. A. Identification, expression, and DNA sequence of the GDP-mannose biosynthesis genes encoded by the O7 rfb gene cluster of strain VW187 (Escherichia coli O7:K1). J Bacteriol. 1993 Jan;175(1):148–158. doi: 10.1128/jb.175.1.148-158.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. McDaniel T. K., Jarvis K. G., Donnenberg M. S., Kaper J. B. A genetic locus of enterocyte effacement conserved among diverse enterobacterial pathogens. Proc Natl Acad Sci U S A. 1995 Feb 28;92(5):1664–1668. doi: 10.1073/pnas.92.5.1664. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Nelson K., Selander R. K. Intergeneric transfer and recombination of the 6-phosphogluconate dehydrogenase gene (gnd) in enteric bacteria. Proc Natl Acad Sci U S A. 1994 Oct 11;91(21):10227–10231. doi: 10.1073/pnas.91.21.10227. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Ochman H., Selander R. K. Standard reference strains of Escherichia coli from natural populations. J Bacteriol. 1984 Feb;157(2):690–693. doi: 10.1128/jb.157.2.690-693.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Perry M. B., MacLean L., Griffith D. W. Structure of the O-chain polysaccharide of the phenol-phase soluble lipopolysaccharide of Escherichia coli 0:157:H7. Biochem Cell Biol. 1986 Jan;64(1):21–28. doi: 10.1139/o86-004. [DOI] [PubMed] [Google Scholar]
  24. Pierson D. E. Mutations affecting lipopolysaccharide enhance ail-mediated entry of Yersinia enterocolitica into mammalian cells. J Bacteriol. 1994 Jul;176(13):4043–4051. doi: 10.1128/jb.176.13.4043-4051.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Reeves P. Evolution of Salmonella O antigen variation by interspecific gene transfer on a large scale. Trends Genet. 1993 Jan;9(1):17–22. doi: 10.1016/0168-9525(93)90067-R. [DOI] [PubMed] [Google Scholar]
  26. Riley L. W., Junio L. N., Libaek L. B., Schoolnik G. K. Plasmid-encoded expression of lipopolysaccharide O-antigenic polysaccharide in enteropathogenic Escherichia coli. Infect Immun. 1987 Sep;55(9):2052–2056. doi: 10.1128/iai.55.9.2052-2056.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Samadpour M., Grimm L. M., Desai B., Alfi D., Ongerth J. E., Tarr P. I. Molecular epidemiology of Escherichia coli O157:H7 strains by bacteriophage lambda restriction fragment length polymorphism analysis: application to a multistate foodborne outbreak and a day-care center cluster. J Clin Microbiol. 1993 Dec;31(12):3179–3183. doi: 10.1128/jcm.31.12.3179-3183.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Stevenson G., Neal B., Liu D., Hobbs M., Packer N. H., Batley M., Redmond J. W., Lindquist L., Reeves P. Structure of the O antigen of Escherichia coli K-12 and the sequence of its rfb gene cluster. J Bacteriol. 1994 Jul;176(13):4144–4156. doi: 10.1128/jb.176.13.4144-4156.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Strockbine N. A., Marques L. R., Newland J. W., Smith H. W., Holmes R. K., O'Brien A. D. Two toxin-converting phages from Escherichia coli O157:H7 strain 933 encode antigenically distinct toxins with similar biologic activities. Infect Immun. 1986 Jul;53(1):135–140. doi: 10.1128/iai.53.1.135-140.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Stroeher U. H., Karageorgos L. E., Morona R., Manning P. A. Serotype conversion in Vibrio cholerae O1. Proc Natl Acad Sci U S A. 1992 Apr 1;89(7):2566–2570. doi: 10.1073/pnas.89.7.2566. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Tarr P. I. Escherichia coli O157:H7: clinical, diagnostic, and epidemiological aspects of human infection. Clin Infect Dis. 1995 Jan;20(1):1–10. doi: 10.1093/clinids/20.1.1. [DOI] [PubMed] [Google Scholar]
  33. Tarr P. I., Neill M. A., Clausen C. R., Newland J. W., Neill R. J., Moseley S. L. Genotypic variation in pathogenic Escherichia coli O157:H7 isolated from patients in Washington, 1984-1987. J Infect Dis. 1989 Feb;159(2):344–347. doi: 10.1093/infdis/159.2.344. [DOI] [PubMed] [Google Scholar]
  34. Taylor R. K., Manoil C., Mekalanos J. J. Broad-host-range vectors for delivery of TnphoA: use in genetic analysis of secreted virulence determinants of Vibrio cholerae. J Bacteriol. 1989 Apr;171(4):1870–1878. doi: 10.1128/jb.171.4.1870-1878.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Tsai C. M., Frasch C. E. A sensitive silver stain for detecting lipopolysaccharides in polyacrylamide gels. Anal Biochem. 1982 Jan 1;119(1):115–119. doi: 10.1016/0003-2697(82)90673-x. [DOI] [PubMed] [Google Scholar]
  36. Voorhis D. L., Dillon S., Formal S. B., Isberg R. R. An O antigen can interfere with the function of the Yersinia pseudotuberculosis invasin protein. Mol Microbiol. 1991 Feb;5(2):317–325. doi: 10.1111/j.1365-2958.1991.tb02112.x. [DOI] [PubMed] [Google Scholar]
  37. Wells J. G., Davis B. R., Wachsmuth I. K., Riley L. W., Remis R. S., Sokolow R., Morris G. K. Laboratory investigation of hemorrhagic colitis outbreaks associated with a rare Escherichia coli serotype. J Clin Microbiol. 1983 Sep;18(3):512–520. doi: 10.1128/jcm.18.3.512-520.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Whittam T. S., Wolfe M. L., Wachsmuth I. K., Orskov F., Orskov I., Wilson R. A. Clonal relationships among Escherichia coli strains that cause hemorrhagic colitis and infantile diarrhea. Infect Immun. 1993 May;61(5):1619–1629. doi: 10.1128/iai.61.5.1619-1629.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Woodall L. D., Russell P. W., Harris S. L., Orndorff P. E. Rapid, synchronous, and stable induction of type 1 piliation in Escherichia coli by using a chromosomal lacUV5 promoter. J Bacteriol. 1993 May;175(9):2770–2778. doi: 10.1128/jb.175.9.2770-2778.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. van der Ley P., Kuipers O., Tommassen J., Lugtenberg B. O-antigenic chains of lipopolysaccharide prevent binding of antibody molecules to an outer membrane pore protein in Enterobacteriaceae. Microb Pathog. 1986 Feb;1(1):43–49. doi: 10.1016/0882-4010(86)90030-6. [DOI] [PubMed] [Google Scholar]
  41. von Heijne G. A new method for predicting signal sequence cleavage sites. Nucleic Acids Res. 1986 Jun 11;14(11):4683–4690. doi: 10.1093/nar/14.11.4683. [DOI] [PMC free article] [PubMed] [Google Scholar]

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