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. 1995 Nov;63(11):4323–4328. doi: 10.1128/iai.63.11.4323-4328.1995

Molecular characterization of a carboxy-terminal eukaryotic-cell-binding domain of intimin from enteropathogenic Escherichia coli.

G Frankel 1, D C Candy 1, E Fabiani 1, J Adu-Bobie 1, S Gil 1, M Novakova 1, A D Phillips 1, G Dougan 1
PMCID: PMC173615  PMID: 7591066

Abstract

A eukaryotic cell-binding domain from the intimin (Int) polypeptide of enteropathogenic Escherichia coli O127 (EPEC) was investigated. Derivatives of the carboxy-terminal 280-amino-acid domains of Int (Int-EPEC280) and the Int homolog invasin (Inv) from Yersinia pseudotuberculosis (InvYP280) were fused to the E. coli maltose-binding protein (MBP), expressed, and purified. The smallest MBP-IntEPEC fusion protein that efficiently mediated binding to HEp-2 cells, monitored by using purified fusion proteins in fluorescence activated cell sorter analysis or by using fluorescent Covaspheres coated with purified fusions, contained the carboxy-terminal 150 amino acids of Int. Replacement of Cys-937 with Ser (IntEPEC280CS) destroyed the cell-binding activity of IntEPEC280. Covaspheres coated with MBP-IntEPEC280 were associated with HEp-2 cell microvilli but failed to induce actin accumulation underneath bound particles or cell spreading on coated plastic surfaces. MBP-IntEPEC280, but not MBP, MBP-IntEPEC280CS, or MBP-InvYP280, inhibited EPEC entry into HEp-2 cells.

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Selected References

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  1. Donnenberg M. S., Kaper J. B. Construction of an eae deletion mutant of enteropathogenic Escherichia coli by using a positive-selection suicide vector. Infect Immun. 1991 Dec;59(12):4310–4317. doi: 10.1128/iai.59.12.4310-4317.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Donnenberg M. S., Tacket C. O., James S. P., Losonsky G., Nataro J. P., Wasserman S. S., Kaper J. B., Levine M. M. Role of the eaeA gene in experimental enteropathogenic Escherichia coli infection. J Clin Invest. 1993 Sep;92(3):1412–1417. doi: 10.1172/JCI116717. [DOI] [PMC free article] [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. Donnenberg M. S., Yu J., Kaper J. B. A second chromosomal gene necessary for intimate attachment of enteropathogenic Escherichia coli to epithelial cells. J Bacteriol. 1993 Aug;175(15):4670–4680. doi: 10.1128/jb.175.15.4670-4680.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Finlay B. B., Rosenshine I., Donnenberg M. S., Kaper J. B. Cytoskeletal composition of attaching and effacing lesions associated with enteropathogenic Escherichia coli adherence to HeLa cells. Infect Immun. 1992 Jun;60(6):2541–2543. doi: 10.1128/iai.60.6.2541-2543.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Francis C. L., Jerse A. E., Kaper J. B., Falkow S. Characterization of interactions of enteropathogenic Escherichia coli O127:H6 with mammalian cells in vitro. J Infect Dis. 1991 Oct;164(4):693–703. doi: 10.1093/infdis/164.4.693. [DOI] [PubMed] [Google Scholar]
  7. 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]
  8. 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]
  9. 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]
  10. Jerse A. E., Kaper J. B. 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. 1991 Dec;59(12):4302–4309. doi: 10.1128/iai.59.12.4302-4309.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. 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]
  12. Knutton S., Baldwin T., Williams P. H., McNeish A. S. Actin accumulation at sites of bacterial adhesion to tissue culture cells: basis of a new diagnostic test for enteropathogenic and enterohemorrhagic Escherichia coli. Infect Immun. 1989 Apr;57(4):1290–1298. doi: 10.1128/iai.57.4.1290-1298.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. 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]
  14. 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]
  15. Levine M. M. Escherichia coli that cause diarrhea: enterotoxigenic, enteropathogenic, enteroinvasive, enterohemorrhagic, and enteroadherent. J Infect Dis. 1987 Mar;155(3):377–389. doi: 10.1093/infdis/155.3.377. [DOI] [PubMed] [Google Scholar]
  16. Moon H. W., Whipp S. C., Argenzio R. A., Levine M. M., Giannella R. A. Attaching and effacing activities of rabbit and human enteropathogenic Escherichia coli in pig and rabbit intestines. Infect Immun. 1983 Sep;41(3):1340–1351. doi: 10.1128/iai.41.3.1340-1351.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Mullis K. B., Faloona F. A. Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction. Methods Enzymol. 1987;155:335–350. doi: 10.1016/0076-6879(87)55023-6. [DOI] [PubMed] [Google Scholar]
  18. Rosenshine I., Donnenberg M. S., Kaper J. B., Finlay B. B. Signal transduction between enteropathogenic Escherichia coli (EPEC) and epithelial cells: EPEC induces tyrosine phosphorylation of host cell proteins to initiate cytoskeletal rearrangement and bacterial uptake. EMBO J. 1992 Oct;11(10):3551–3560. doi: 10.1002/j.1460-2075.1992.tb05438.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Scaletsky I. C., Silva M. L., Trabulsi L. R. Distinctive patterns of adherence of enteropathogenic Escherichia coli to HeLa cells. Infect Immun. 1984 Aug;45(2):534–536. doi: 10.1128/iai.45.2.534-536.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. 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]
  21. Tran Van Nhieu G., Isberg R. R. Bacterial internalization mediated by beta 1 chain integrins is determined by ligand affinity and receptor density. EMBO J. 1993 May;12(5):1887–1895. doi: 10.1002/j.1460-2075.1993.tb05837.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Ulshen M. H., Rollo J. L. Pathogenesis of escherichia coli gastroenteritis in man--another mechanism. N Engl J Med. 1980 Jan 10;302(2):99–101. doi: 10.1056/NEJM198001103020207. [DOI] [PubMed] [Google Scholar]
  23. Van Nhieu G. T., Isberg R. R. The Yersinia pseudotuberculosis invasin protein and human fibronectin bind to mutually exclusive sites on the alpha 5 beta 1 integrin receptor. J Biol Chem. 1991 Dec 25;266(36):24367–24375. [PubMed] [Google Scholar]
  24. 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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