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. 1997 Jul;65(7):2528–2536. doi: 10.1128/iai.65.7.2528-2536.1997

Intimin-dependent binding of enteropathogenic Escherichia coli to host cells triggers novel signaling events, including tyrosine phosphorylation of phospholipase C-gamma1.

B Kenny 1, B B Finlay 1
PMCID: PMC175357  PMID: 9199415

Abstract

Enteropathogenic Escherichia coli (EPEC) interactions with HeLa epithelial cells induced the tyrosine phosphorylation of a host protein of approximately 150 kDa, Hp150. Phosphorylation of this protein band was dependent on the interaction of the EPEC protein intimin with epithelial cell surfaces and was correlated with pedestal formation. Hp150 phosphorylation was specifically inhibited by the addition of cytochalasin D, an inhibitor of actin polymerization, although this appeared to be an indirect effect preventing interaction of intimin with its receptor, tyrosine-phosphorylated Hp90, and thus triggering Hp150 phosphorylation. This suggests the involvement of an actin-based movement of membrane-bound tyrosine-phosphorylated Hp90 to allow its interaction with intimin. Analysis of the tyrosine-phosphorylated Hp150 protein demonstrated that it is heterogeneous in composition, with phospholipase C-gamma1 (PLC-gamma1) being a minor component. Activation of PLC-gamma1 by tyrosine phosphorylation leads to inositol triphosphate and Ca2+ fluxes, events detected following EPEC infection. EPEC also induced tyrosine dephosphorylation of host proteins, including a 240-kDa host protein (Hp240), following EPEC infection. Protein dephosphorylation appears to be a signaling event which occurs independently of intimin. Inhibition of host tyrosine dephosphorylation events by the addition of the tyrosine phosphatase inhibitor sodium vanadate did not prevent actin accumulation beneath the adherent bacteria. We conclude that EPEC induces two sets of signaling events following infection. One set is dependent on EPEC proteins secreted by the type III secretion pathway (EspA and EspB) which induces Hp90 tyrosine phosphorylation and dephosphorylation of host phosphotyrosine proteins. The second set, which is also dependent on the first signaling events, requires intimin interaction with its receptor, tyrosine-phosphorylated Hp90, to trigger Hp150 and PLC-gamma1 tyrosine phosphorylation as well as pedestal formation. Inhibition of pedestal formation by tyrosine kinase inhibitors indicates an important role for tyrosine phosphorylation events during EPEC subversion of host processes.

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Selected References

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  1. Baldwin T. J., Ward W., Aitken A., Knutton S., Williams P. H. Elevation of intracellular free calcium levels in HEp-2 cells infected with enteropathogenic Escherichia coli. Infect Immun. 1991 May;59(5):1599–1604. doi: 10.1128/iai.59.5.1599-1604.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Berridge M. J. Inositol trisphosphate and calcium signalling. Nature. 1993 Jan 28;361(6410):315–325. doi: 10.1038/361315a0. [DOI] [PubMed] [Google Scholar]
  3. Bliska J. B., Guan K. L., Dixon J. E., Falkow S. Tyrosine phosphate hydrolysis of host proteins by an essential Yersinia virulence determinant. Proc Natl Acad Sci U S A. 1991 Feb 15;88(4):1187–1191. doi: 10.1073/pnas.88.4.1187. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Donnenberg M. S., Calderwood S. B., Donohue-Rolfe A., Keusch G. T., Kaper J. B. Construction and analysis of TnphoA mutants of enteropathogenic Escherichia coli unable to invade HEp-2 cells. Infect Immun. 1990 Jun;58(6):1565–1571. doi: 10.1128/iai.58.6.1565-1571.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Donnenberg M. S., Girón J. A., Nataro J. P., Kaper J. B. A plasmid-encoded type IV fimbrial gene of enteropathogenic Escherichia coli associated with localized adherence. Mol Microbiol. 1992 Nov;6(22):3427–3437. doi: 10.1111/j.1365-2958.1992.tb02210.x. [DOI] [PubMed] [Google Scholar]
  6. 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]
  7. 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]
  8. Dytoc M., Fedorko L., Sherman P. M. Signal transduction in human epithelial cells infected with attaching and effacing Escherichia coli in vitro. Gastroenterology. 1994 May;106(5):1150–1161. doi: 10.1016/0016-5085(94)90004-3. [DOI] [PubMed] [Google Scholar]
  9. 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]
  10. Foubister V., Rosenshine I., Donnenberg M. S., Finlay B. B. The eaeB gene of enteropathogenic Escherichia coli is necessary for signal transduction in epithelial cells. Infect Immun. 1994 Jul;62(7):3038–3040. doi: 10.1128/iai.62.7.3038-3040.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Foubister V., Rosenshine I., Finlay B. B. A diarrheal pathogen, enteropathogenic Escherichia coli (EPEC), triggers a flux of inositol phosphates in infected epithelial cells. J Exp Med. 1994 Mar 1;179(3):993–998. doi: 10.1084/jem.179.3.993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Goldschmidt-Clermont P. J., Kim J. W., Machesky L. M., Rhee S. G., Pollard T. D. Regulation of phospholipase C-gamma 1 by profilin and tyrosine phosphorylation. Science. 1991 Mar 8;251(4998):1231–1233. doi: 10.1126/science.1848725. [DOI] [PubMed] [Google Scholar]
  13. 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]
  14. Jarvis K. G., Girón J. A., Jerse A. E., McDaniel T. K., Donnenberg M. S., Kaper J. B. Enteropathogenic Escherichia coli contains a putative type III secretion system necessary for the export of proteins involved in attaching and effacing lesion formation. Proc Natl Acad Sci U S A. 1995 Aug 15;92(17):7996–8000. doi: 10.1073/pnas.92.17.7996. [DOI] [PMC free article] [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. Kenny B., Finlay B. B. Protein secretion by enteropathogenic Escherichia coli is essential for transducing signals to epithelial cells. Proc Natl Acad Sci U S A. 1995 Aug 15;92(17):7991–7995. doi: 10.1073/pnas.92.17.7991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kenny B., Lai L. C., Finlay B. B., Donnenberg M. S. EspA, a protein secreted by enteropathogenic Escherichia coli, is required to induce signals in epithelial cells. Mol Microbiol. 1996 Apr;20(2):313–323. doi: 10.1111/j.1365-2958.1996.tb02619.x. [DOI] [PubMed] [Google Scholar]
  18. 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]
  19. Knutton S., Lloyd D. R., McNeish A. S. Adhesion of enteropathogenic Escherichia coli to human intestinal enterocytes and cultured human intestinal mucosa. Infect Immun. 1987 Jan;55(1):69–77. doi: 10.1128/iai.55.1.69-77.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. 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]
  21. Manjarrez-Hernandez H. A., Baldwin T. J., Aitken A., Knutton S., Williams P. H. Intestinal epithelial cell protein phosphorylation in enteropathogenic Escherichia coli diarrhoea. Lancet. 1992 Feb 29;339(8792):521–523. doi: 10.1016/0140-6736(92)90340-9. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. Rijnboutt S., Aerts H. M., Geuze H. J., Tager J. M., Strous G. J. Mannose 6-phosphate-independent membrane association of cathepsin D, glucocerebrosidase, and sphingolipid-activating protein in HepG2 cells. J Biol Chem. 1991 Mar 15;266(8):4862–4868. [PubMed] [Google Scholar]
  24. Robins-Browne R. M. Traditional enteropathogenic Escherichia coli of infantile diarrhea. Rev Infect Dis. 1987 Jan-Feb;9(1):28–53. doi: 10.1093/clinids/9.1.28. [DOI] [PubMed] [Google Scholar]
  25. 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]
  26. Rosenshine I., Ruschkowski S., Finlay B. B. Expression of attaching/effacing activity by enteropathogenic Escherichia coli depends on growth phase, temperature, and protein synthesis upon contact with epithelial cells. Infect Immun. 1996 Mar;64(3):966–973. doi: 10.1128/iai.64.3.966-973.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Rosenshine I., Ruschkowski S., Stein M., Reinscheid D. J., Mills S. D., Finlay B. B. A pathogenic bacterium triggers epithelial signals to form a functional bacterial receptor that mediates actin pseudopod formation. EMBO J. 1996 Jun 3;15(11):2613–2624. [PMC free article] [PubMed] [Google Scholar]
  28. Sultzman L., Ellis C., Lin L. L., Pawson T., Knopf J. Platelet-derived growth factor increases the in vivo activity of phospholipase C-gamma 1 and phospholipase C-gamma 2. Mol Cell Biol. 1991 Apr;11(4):2018–2025. doi: 10.1128/mcb.11.4.2018. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Vuopio-Varkila J., Schoolnik G. K. Localized adherence by enteropathogenic Escherichia coli is an inducible phenotype associated with the expression of new outer membrane proteins. J Exp Med. 1991 Nov 1;174(5):1167–1177. doi: 10.1084/jem.174.5.1167. [DOI] [PMC free article] [PubMed] [Google Scholar]

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