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. 1996 Jun;64(6):2225–2233. doi: 10.1128/iai.64.6.2225-2233.1996

Truncated enterohemorrhagic Escherichia coli (EHEC) O157:H7 intimin (EaeA) fusion proteins promote adherence of EHEC strains to HEp-2 cells.

M L McKee 1, A D O'Brien 1
PMCID: PMC174060  PMID: 8675331

Abstract

Intimin, the product of the eaeA gene in enterohemorrhagic Escherichia coli O157:H7 (EHEC), is required for intimate adherence of these organisms to tissue culture cells and formation of the attaching and effacing lesion in the gnotobiotic pig. Because of the importance of intimin in the pathogenesis of EHEC O157:H7 infection in this animal model, we began a structure-function analysis of EaeA. For this purpose, we constructed amino-terminal fusions of the intimin protein with six histidine residues to form two independent fusions. The longer fusion, RIHisEae, contained 900 of the 935 predicted amino acids and included all but the extreme amino terminus. The second fusion, RVHdHisEae, consisted of the carboxyl two-thirds of the protein. Purified extracts of either construct enhanced binding of wild-type 86-24 to HEp-2 cells and conferred HEp-2 cell adherence on 86-24eaeDelta10, an eaeA deletion mutant, and B2F1, an EHEC O91:1-121 eaeA mutant strain. When 86-24eaeDelta10 was transformed with either of the plasmids encoding the intimin fusion proteins, the transformant behaved like the wild-type parent strain and displayed localized adherence to HEp-2 cells, with positive fluorescent-actin staining. In addition, polyclonal antisera raised against RIHisEae reacted with both fusion constructs and recognized an outer membrane protein of the same mass as intimin (97 kDa) in EHEC and enteropathogenic E. coli but not E. coli K-12. The intimin-specific antisera also blocked adherence of EHEC to HEp-2 cells. Thus, intimin (i) is a 97-kDa outer membrane protein in EHEC that serves as a requisite adhesin for attachment of the bacteria to epithelial cells, even when the protein is truncated by one-third at its amino terminus and (ii) can be added exogenously to specifically facilitate HEp-2 cell adherence of EHEC but not E. coli K-12.

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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. Albert M. J., Faruque S. M., Ansaruzzaman M., Islam M. M., Haider K., Alam K., Kabir I., Robins-Browne R. Sharing of virulence-associated properties at the phenotypic and genetic levels between enteropathogenic Escherichia coli and Hafnia alvei. J Med Microbiol. 1992 Nov;37(5):310–314. doi: 10.1099/00222615-37-5-310. [DOI] [PubMed] [Google Scholar]
  3. Donnenberg M. S., Tzipori S., McKee M. L., O'Brien A. D., Alroy J., Kaper J. B. The role of the eae gene of enterohemorrhagic Escherichia coli in intimate attachment in vitro and in a porcine model. J Clin Invest. 1993 Sep;92(3):1418–1424. doi: 10.1172/JCI116718. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Dytoc M., Soni R., Cockerill F., 3rd, De Azavedo J., Louie M., Brunton J., Sherman P. Multiple determinants of verotoxin-producing Escherichia coli O157:H7 attachment-effacement. Infect Immun. 1993 Aug;61(8):3382–3391. doi: 10.1128/iai.61.8.3382-3391.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Francis D. H., Collins J. E., Duimstra J. R. Infection of gnotobiotic pigs with an Escherichia coli O157:H7 strain associated with an outbreak of hemorrhagic colitis. Infect Immun. 1986 Mar;51(3):953–956. doi: 10.1128/iai.51.3.953-956.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Frankel G., Candy D. C., Everest P., Dougan G. Characterization of the C-terminal domains of intimin-like proteins of enteropathogenic and enterohemorrhagic Escherichia coli, Citrobacter freundii, and Hafnia alvei. Infect Immun. 1994 May;62(5):1835–1842. doi: 10.1128/iai.62.5.1835-1842.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Fratamico P. M., Bhaduri S., Buchanan R. L. Studies on Escherichia coli serotype O157:H7 strains containing a 60-MDa plasmid and on 60-MDa plasmid-cured derivatives. J Med Microbiol. 1993 Nov;39(5):371–381. doi: 10.1099/00222615-39-5-371. [DOI] [PubMed] [Google Scholar]
  8. 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]
  9. Gómez-Duarte O. G., Kaper J. B. A plasmid-encoded regulatory region activates chromosomal eaeA expression in enteropathogenic Escherichia coli. Infect Immun. 1995 May;63(5):1767–1776. doi: 10.1128/iai.63.5.1767-1776.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Isberg R. R., Falkow S. A single genetic locus encoded by Yersinia pseudotuberculosis permits invasion of cultured animal cells by Escherichia coli K-12. Nature. 1985 Sep 19;317(6034):262–264. doi: 10.1038/317262a0. [DOI] [PubMed] [Google Scholar]
  11. Isberg R. R., Leong J. M. Cultured mammalian cells attach to the invasin protein of Yersinia pseudotuberculosis. Proc Natl Acad Sci U S A. 1988 Sep;85(18):6682–6686. doi: 10.1073/pnas.85.18.6682. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Isberg R. R., Leong J. M. Multiple beta 1 chain integrins are receptors for invasin, a protein that promotes bacterial penetration into mammalian cells. Cell. 1990 Mar 9;60(5):861–871. doi: 10.1016/0092-8674(90)90099-z. [DOI] [PubMed] [Google Scholar]
  13. Isberg R. R., Voorhis D. L., Falkow S. Identification of invasin: a protein that allows enteric bacteria to penetrate cultured mammalian cells. Cell. 1987 Aug 28;50(5):769–778. doi: 10.1016/0092-8674(87)90335-7. [DOI] [PubMed] [Google Scholar]
  14. Ito H., Terai A., Kurazono H., Takeda Y., Nishibuchi M. Cloning and nucleotide sequencing of Vero toxin 2 variant genes from Escherichia coli O91:H21 isolated from a patient with the hemolytic uremic syndrome. Microb Pathog. 1990 Jan;8(1):47–60. doi: 10.1016/0882-4010(90)90007-d. [DOI] [PubMed] [Google Scholar]
  15. Jerse A. E., Yu J., Tall B. D., Kaper J. B. 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. 1990 Oct;87(20):7839–7843. doi: 10.1073/pnas.87.20.7839. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Karch H., Heesemann J., Laufs R., O'Brien A. D., Tacket C. O., Levine M. M. A plasmid of enterohemorrhagic Escherichia coli O157:H7 is required for expression of a new fimbrial antigen and for adhesion to epithelial cells. Infect Immun. 1987 Feb;55(2):455–461. doi: 10.1128/iai.55.2.455-461.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  18. Leong J. M., Fournier R. S., Isberg R. R. Identification of the integrin binding domain of the Yersinia pseudotuberculosis invasin protein. EMBO J. 1990 Jun;9(6):1979–1989. doi: 10.1002/j.1460-2075.1990.tb08326.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Leong J. M., Morrissey P. E., Isberg R. R. A 76-amino acid disulfide loop in the Yersinia pseudotuberculosis invasin protein is required for integrin receptor recognition. J Biol Chem. 1993 Sep 25;268(27):20524–20532. [PubMed] [Google Scholar]
  20. Leong J. M., Morrissey P. E., Marra A., Isberg R. R. An aspartate residue of the Yersinia pseudotuberculosis invasin protein that is critical for integrin binding. EMBO J. 1995 Feb 1;14(3):422–431. doi: 10.1002/j.1460-2075.1995.tb07018.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Levine M. M., Bergquist E. J., Nalin D. R., Waterman D. H., Hornick R. B., Young C. R., Sotman S. Escherichia coli strains that cause diarrhoea but do not produce heat-labile or heat-stable enterotoxins and are non-invasive. Lancet. 1978 May 27;1(8074):1119–1122. doi: 10.1016/s0140-6736(78)90299-4. [DOI] [PubMed] [Google Scholar]
  22. Louie M., de Azavedo J. C., Handelsman M. Y., Clark C. G., Ally B., Dytoc M., Sherman P., Brunton J. Expression and characterization of the eaeA gene product of Escherichia coli serotype O157:H7. Infect Immun. 1993 Oct;61(10):4085–4092. doi: 10.1128/iai.61.10.4085-4092.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. 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]
  24. McKee M. L., Melton-Celsa A. R., Moxley R. A., Francis D. H., O'Brien A. D. Enterohemorrhagic Escherichia coli O157:H7 requires intimin to colonize the gnotobiotic pig intestine and to adhere to HEp-2 cells. Infect Immun. 1995 Sep;63(9):3739–3744. doi: 10.1128/iai.63.9.3739-3744.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. McKee M. L., O'Brien A. D. Investigation of enterohemorrhagic Escherichia coli O157:H7 adherence characteristics and invasion potential reveals a new attachment pattern shared by intestinal E. coli. Infect Immun. 1995 May;63(5):2070–2074. doi: 10.1128/iai.63.5.2070-2074.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Meier-Dieter U., Acker G., Mayer H. Detection of enterobacterial common antigen on bacterial cell surfaces by colony-immunoblotting: effect of its linkage to lipopolysaccharide. FEMS Microbiol Lett. 1989 May;50(1-2):215–219. doi: 10.1016/0378-1097(89)90489-8. [DOI] [PubMed] [Google Scholar]
  27. Meier-Dieter U., Barr K., Starman R., Hatch L., Rick P. D. Nucleotide sequence of the Escherichia coli rfe gene involved in the synthesis of enterobacterial common antigen. Molecular cloning of the rfe-rff gene cluster. J Biol Chem. 1992 Jan 15;267(2):746–753. [PubMed] [Google Scholar]
  28. O'Brien A. D., Holmes R. K. Shiga and Shiga-like toxins. Microbiol Rev. 1987 Jun;51(2):206–220. doi: 10.1128/mr.51.2.206-220.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Pepe J. C., Miller V. L. Yersinia enterocolitica invasin: a primary role in the initiation of infection. Proc Natl Acad Sci U S A. 1993 Jul 15;90(14):6473–6477. doi: 10.1073/pnas.90.14.6473. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Potter M. E., Kaufmann A. F., Thomason B. M., Blake P. A., Farmer J. J., 3rd Diarrhea due to Escherichia coli O157:H7 in the infant rabbit. J Infect Dis. 1985 Dec;152(6):1341–1343. doi: 10.1093/infdis/152.6.1341. [DOI] [PubMed] [Google Scholar]
  31. Riley L. W., Remis R. S., Helgerson S. D., McGee H. B., Wells J. G., Davis B. R., Hebert R. J., Olcott E. S., Johnson L. M., Hargrett N. T. Hemorrhagic colitis associated with a rare Escherichia coli serotype. N Engl J Med. 1983 Mar 24;308(12):681–685. doi: 10.1056/NEJM198303243081203. [DOI] [PubMed] [Google Scholar]
  32. Riley L. W. The epidemiologic, clinical, and microbiologic features of hemorrhagic colitis. Annu Rev Microbiol. 1987;41:383–407. doi: 10.1146/annurev.mi.41.100187.002123. [DOI] [PubMed] [Google Scholar]
  33. Schauer D. B., Falkow S. Attaching and effacing locus of a Citrobacter freundii biotype that causes transmissible murine colonic hyperplasia. Infect Immun. 1993 Jun;61(6):2486–2492. doi: 10.1128/iai.61.6.2486-2492.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Schauer D. B., Falkow S. The eae gene of Citrobacter freundii biotype 4280 is necessary for colonization in transmissible murine colonic hyperplasia. Infect Immun. 1993 Nov;61(11):4654–4661. doi: 10.1128/iai.61.11.4654-4661.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Schmitt C. K., Darnell S. C., Tesh V. L., Stocker B. A., O'Brien A. D. Mutation of flgM attenuates virulence of Salmonella typhimurium, and mutation of fliA represses the attenuated phenotype. J Bacteriol. 1994 Jan;176(2):368–377. doi: 10.1128/jb.176.2.368-377.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Sherman P. M., Soni R. Adherence of Vero cytotoxin-producing Escherichia coli of serotype O157:H7 to human epithelial cells in tissue culture: role of outer membranes as bacterial adhesins. J Med Microbiol. 1988 May;26(1):11–17. doi: 10.1099/00222615-26-1-11. [DOI] [PubMed] [Google Scholar]
  37. Sherman P., Cockerill F., 3rd, Soni R., Brunton J. Outer membranes are competitive inhibitors of Escherichia coli O157:H7 adherence to epithelial cells. Infect Immun. 1991 Mar;59(3):890–899. doi: 10.1128/iai.59.3.890-899.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Sizemore D. R., Fink P. S., Ou J. T., Baron L., Kopecko D. J., Warren R. L. Tn5 mutagenesis of the Salmonella typhimurium 100 kb plasmid: definition of new virulence regions. Microb Pathog. 1991 Jun;10(6):493–499. doi: 10.1016/0882-4010(91)90116-r. [DOI] [PubMed] [Google Scholar]
  39. Tabor S., Richardson C. C. A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes. Proc Natl Acad Sci U S A. 1985 Feb;82(4):1074–1078. doi: 10.1073/pnas.82.4.1074. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. 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]
  41. Tesh V. L., O'Brien A. D. The pathogenic mechanisms of Shiga toxin and the Shiga-like toxins. Mol Microbiol. 1991 Aug;5(8):1817–1822. doi: 10.1111/j.1365-2958.1991.tb00805.x. [DOI] [PubMed] [Google Scholar]
  42. Toth I., Cohen M. L., Rumschlag H. S., Riley L. W., White E. H., Carr J. H., Bond W. W., Wachsmuth I. K. Influence of the 60-megadalton plasmid on adherence of Escherichia coli O157:H7 and genetic derivatives. Infect Immun. 1990 May;58(5):1223–1231. doi: 10.1128/iai.58.5.1223-1231.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Tzipori S., Gibson R., Montanaro J. Nature and distribution of mucosal lesions associated with enteropathogenic and enterohemorrhagic Escherichia coli in piglets and the role of plasmid-mediated factors. Infect Immun. 1989 Apr;57(4):1142–1150. doi: 10.1128/iai.57.4.1142-1150.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Wadolkowski E. A., Burris J. A., O'Brien A. D. Mouse model for colonization and disease caused by enterohemorrhagic Escherichia coli O157:H7. Infect Immun. 1990 Aug;58(8):2438–2445. doi: 10.1128/iai.58.8.2438-2445.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Yu J., Kaper J. B. Cloning and characterization of the eae gene of enterohaemorrhagic Escherichia coli O157:H7. Mol Microbiol. 1992 Feb;6(3):411–417. doi: 10.1111/j.1365-2958.1992.tb01484.x. [DOI] [PubMed] [Google Scholar]

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