Skip to main content
PMC home page
Infection and Immunity logoLink to Infection and Immunity
. 1997 Aug;65(8):3277–3285. doi: 10.1128/iai.65.8.3277-3285.1997

Activation of host cell protein kinase C by enteropathogenic Escherichia coli.

J K Crane 1, J S Oh 1
PMCID: PMC175464  PMID: 9234787

Abstract

Enteropathogenic Escherichia coli (EPEC) consists of a group of diarrhea-producing E. coli strains, common in developing countries, which do not produce classical toxins and are not truly invasive. EPEC strains adhere to mammalian cells in an intimate fashion, trigger a localized increase in intracellular calcium levels, and elevate inositol phosphate production. We hypothesized that these mediators could activate host cell protein kinase C (PKC) and tested this idea in vitro with two cultured human cell lines, HeLa cells and T84 cells. Using a recently described subculturing protocol to "induce" or accelerate EPEC adherence, we infected the cells with EPEC at a multiplicity of infection of approximately 100:1 for 30 to 60 min. Under these conditions, EPEC E2348 increased membrane-bound PKC activity 1.5- to 2.3-fold in HeLa cells and T84 cells, respectively. The increase in membrane-bound PKC activity was accompanied by a decrease in cytosolic PKC activity in EPEC-infected HeLa cells. Nonadherent laboratory E. coli strains such as HB101 and H.S. failed to trigger any consistent change in PKC production, similar to the nonadherent mutant strains derived from E2348, JPN15 (plasmid cured) and CVD206 (eaeA). In addition, immunoblots performed on extracts of T84 cells with a monoclonal antibody against PKC-alpha showed an increased PKC content in membranes of EPEC-infected cells. Finally, EPEC-infected T84 cells showed a 60% increase in responsiveness to the E. coli heat-stable toxin. We conclude that mediators produced in response to EPEC adherence activate PKC in intestinal and nonintestinal cells.

Full Text

The Full Text of this article is available as a PDF (2.4 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Baldwin T. J., Brooks S. F., Knutton S., Manjarrez Hernandez H. A., Aitken A., Williams P. H. Protein phosphorylation by protein kinase C in HEp-2 cells infected with enteropathogenic Escherichia coli. Infect Immun. 1990 Mar;58(3):761–765. doi: 10.1128/iai.58.3.761-765.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Baldwin T. J., Knutton S., Haigh R., Williams P. H., Palmer H. M., Aitken A., Borriello S. P. Hijacking host cell signal transduction mechanisms during infection with enteropathogenic Escherichia coli. Biochem Soc Trans. 1996 May;24(2):552–558. doi: 10.1042/bst0240552. [DOI] [PubMed] [Google Scholar]
  3. Baldwin T. J., Lee-Delaunay M. B., Knutton S., Williams P. H. Calcium-calmodulin dependence of actin accretion and lethality in cultured HEp-2 cells infected with enteropathogenic Escherichia coli. Infect Immun. 1993 Feb;61(2):760–763. doi: 10.1128/iai.61.2.760-763.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. 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]
  5. Berger S. A., Rowan K., Morrison H. D., Ziltener H. J. Identification of a bacterial inhibitor of protein kinases. Mechanism and role in host cell invasion. J Biol Chem. 1996 Sep 20;271(38):23431–23437. doi: 10.1074/jbc.271.38.23431. [DOI] [PubMed] [Google Scholar]
  6. Beubler E., Schirgi-Degen A. Stimulation of enterocyte protein kinase C by laxatives in-vitro. J Pharm Pharmacol. 1993 Jan;45(1):59–62. doi: 10.1111/j.2042-7158.1993.tb03680.x. [DOI] [PubMed] [Google Scholar]
  7. Chang E. B., Wang N. S., Rao M. C. Phorbol ester stimulation of active anion secretion in intestine. Am J Physiol. 1985 Sep;249(3 Pt 1):C356–C361. doi: 10.1152/ajpcell.1985.249.3.C356. [DOI] [PubMed] [Google Scholar]
  8. Chen S. G., Murakami K. Synergistic activation of type III protein kinase C by cis-fatty acid and diacylglycerol. Biochem J. 1992 Feb 15;282(Pt 1):33–39. doi: 10.1042/bj2820033. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Crane J. K., Burrell L. L., Weikel C. S., Guerrant R. L. Carbachol mimics phorbol esters in its ability to enhance cyclic GMP production by STa, the heat-stable toxin of Escherichia coli. FEBS Lett. 1990 Nov 12;274(1-2):199–202. doi: 10.1016/0014-5793(90)81363-s. [DOI] [PubMed] [Google Scholar]
  10. Crane J. K., Shanks K. L. Phosphorylation and activation of the intestinal guanylyl cyclase receptor for Escherichia coli heat-stable toxin by protein kinase C. Mol Cell Biochem. 1996 Dec 20;165(2):111–120. doi: 10.1007/BF00229472. [DOI] [PubMed] [Google Scholar]
  11. Crane J. K., Wehner M. S., Bolen E. J., Sando J. J., Linden J., Guerrant R. L., Sears C. L. Regulation of intestinal guanylate cyclase by the heat-stable enterotoxin of Escherichia coli (STa) and protein kinase C. Infect Immun. 1992 Dec;60(12):5004–5012. doi: 10.1128/iai.60.12.5004-5012.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. 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]
  13. Donnenberg M. S., Kaper J. B. Enteropathogenic Escherichia coli. Infect Immun. 1992 Oct;60(10):3953–3961. doi: 10.1128/iai.60.10.3953-3961.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Donnenberg M. S., Nataro J. P. Methods for studying adhesion of diarrheagenic Escherichia coli. Methods Enzymol. 1995;253:324–336. doi: 10.1016/s0076-6879(95)53028-2. [DOI] [PubMed] [Google Scholar]
  15. Donowitz M., Montgomery J. L., Walker M. S., Cohen M. E. Brush-border tyrosine phosphorylation stimulates ileal neutral NaCl absorption and brush-border Na(+)-H+ exchange. Am J Physiol. 1994 Apr;266(4 Pt 1):G647–G656. doi: 10.1152/ajpgi.1994.266.4.G647. [DOI] [PubMed] [Google Scholar]
  16. 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]
  17. Fasano A., Fiorentini C., Donelli G., Uzzau S., Kaper J. B., Margaretten K., Ding X., Guandalini S., Comstock L., Goldblum S. E. Zonula occludens toxin modulates tight junctions through protein kinase C-dependent actin reorganization, in vitro. J Clin Invest. 1995 Aug;96(2):710–720. doi: 10.1172/JCI118114. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Fondacaro J. D., Henderson L. S. Evidence for protein kinase C as a regulator of intestinal electrolyte transport. Am J Physiol. 1985 Sep;249(3 Pt 1):G422–G426. doi: 10.1152/ajpgi.1985.249.3.G422. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. Hecht G., Robinson B., Koutsouris A. Reversible disassembly of an intestinal epithelial monolayer by prolonged exposure to phorbol ester. Am J Physiol. 1994 Feb;266(2 Pt 1):G214–G221. doi: 10.1152/ajpgi.1994.266.2.G214. [DOI] [PubMed] [Google Scholar]
  21. Koumi S., Sato R., Aramaki T. Activation of the plasma membrane chloride channel by protein kinase C in isolated guinea-pig hepatocytes. J Physiol. 1995 Sep 1;487(Pt 2):379–394. doi: 10.1113/jphysiol.1995.sp020887. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Law D. Adhesion and its role in the virulence of enteropathogenic Escherichia coli. Clin Microbiol Rev. 1994 Apr;7(2):152–173. doi: 10.1128/cmr.7.2.152. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. 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]
  24. Levine M. M., Nataro J. P., Karch H., Baldini M. M., Kaper J. B., Black R. E., Clements M. L., O'Brien A. D. The diarrheal response of humans to some classic serotypes of enteropathogenic Escherichia coli is dependent on a plasmid encoding an enteroadhesiveness factor. J Infect Dis. 1985 Sep;152(3):550–559. doi: 10.1093/infdis/152.3.550. [DOI] [PubMed] [Google Scholar]
  25. Levine S. A., Donowitz M., Watson A. J., Sharp G. W., Crane J. K., Weikel C. S. Characterization of the synergistic interaction of Escherichia coli heat-stable toxin and carbachol. Am J Physiol. 1991 Oct;261(4 Pt 1):G592–G601. doi: 10.1152/ajpgi.1991.261.4.G592. [DOI] [PubMed] [Google Scholar]
  26. 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]
  27. Mathewson J. J., Oberhelman R. A., Dupont H. L., Javier de la Cabada F., Garibay E. V. Enteroadherent Escherichia coli as a cause of diarrhea among children in Mexico. J Clin Microbiol. 1987 Oct;25(10):1917–1919. doi: 10.1128/jcm.25.10.1917-1919.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Philpott D. J., McKay D. M., Sherman P. M., Perdue M. H. Infection of T84 cells with enteropathogenic Escherichia coli alters barrier and transport functions. Am J Physiol. 1996 Apr;270(4 Pt 1):G634–G645. doi: 10.1152/ajpgi.1996.270.4.G634. [DOI] [PubMed] [Google Scholar]
  29. Picciotto M. R., Cohn J. A., Bertuzzi G., Greengard P., Nairn A. C. Phosphorylation of the cystic fibrosis transmembrane conductance regulator. J Biol Chem. 1992 Jun 25;267(18):12742–12752. [PubMed] [Google Scholar]
  30. 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]
  31. 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]
  32. 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]
  33. Rothbaum R. J., Partin J. C., Saalfield K., McAdams A. J. An ultrastructural study of enteropathogenic Escherichia coli infection in human infants. Ultrastruct Pathol. 1983 Jun;4(4):291–304. doi: 10.3109/01913128309140582. [DOI] [PubMed] [Google Scholar]
  34. Rothbaum R., McAdams A. J., Giannella R., Partin J. C. A clinicopathologic study of enterocyte-adherent Escherichia coli: a cause of protracted diarrhea in infants. Gastroenterology. 1982 Aug;83(2):441–454. [PubMed] [Google Scholar]
  35. Savarino S. J., Fasano A., Robertson D. C., Levine M. M. Enteroaggregative Escherichia coli elaborate a heat-stable enterotoxin demonstrable in an in vitro rabbit intestinal model. J Clin Invest. 1991 Apr;87(4):1450–1455. doi: 10.1172/JCI115151. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Savarino S. J., McVeigh A., Watson J., Cravioto A., Molina J., Echeverria P., Bhan M. K., Levine M. M., Fasano A. Enteroaggregative Escherichia coli heat-stable enterotoxin is not restricted to enteroaggregative E. coli. J Infect Dis. 1996 Apr;173(4):1019–1022. doi: 10.1093/infdis/173.4.1019. [DOI] [PubMed] [Google Scholar]
  37. Sears C. L., Kaper J. B. Enteric bacterial toxins: mechanisms of action and linkage to intestinal secretion. Microbiol Rev. 1996 Mar;60(1):167–215. doi: 10.1128/mr.60.1.167-215.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Spitz J., Yuhan R., Koutsouris A., Blatt C., Alverdy J., Hecht G. Enteropathogenic Escherichia coli adherence to intestinal epithelial monolayers diminishes barrier function. Am J Physiol. 1995 Feb;268(2 Pt 1):G374–G379. doi: 10.1152/ajpgi.1995.268.2.G374. [DOI] [PubMed] [Google Scholar]
  39. 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]
  40. Walker J. M., Sando J. J. Activation of protein kinase C by short chain phosphatidylcholines. J Biol Chem. 1988 Apr 5;263(10):4537–4540. [PubMed] [Google Scholar]
  41. Weikel C. S., Sando J. J., Guerrant R. L. Stimulation of porcine jejunal ion secretion in vivo by protein kinase-C activators. J Clin Invest. 1985 Dec;76(6):2430–2435. doi: 10.1172/JCI112258. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Weikel C. S., Spann C. L., Chambers C. P., Crane J. K., Linden J., Hewlett E. L. Phorbol esters enhance the cyclic GMP response of T84 cells to the heat-stable enterotoxin of Escherichia coli (STa). Infect Immun. 1990 May;58(5):1402–1407. doi: 10.1128/iai.58.5.1402-1407.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Infection and Immunity are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES