, Volume 28, Issue 6, pp 345–353

Enteropathogenic Escherichia coli Outer Membrane Proteins Induce iNOS by Activation of NF-κB and MAP Kinases

  • Vasantha Malladi
  • Manjula Puthenedam
  • Peter H. Williams
  • Arun Balakrishnan


Enteropathogenic Escherichia coli (EPEC) infects the human intestinal epithelium and is a major cause of infantile diarrhea in developing countries. Nitric oxide (NO) is an important modulator of intestinal inflammatory response. The aim of the present study was to investigate whether EPEC outer membrane proteins (OMPs) up regulate epithelial cell expression of inducible nitric oxide synthase (iNOS) and to examine the role of NF-κB and MAP kinases (MAPK) on nitrite production. iNOS mRNA expression was assessed by RT-PCR. Nitrite levels were measured by Griess reaction. NF-κB activation by OMPs was evaluated by EMSA and immunoblotting was done to detect MAPK activation. EPEC OMP up regulated iNOS, induced nitrite production and NF-κB and MAPK were activated in caco-2 cells. The nitrite levels decreased when NF-κB and MAPK inhibitors were used. Thus, EPEC OMPs induce iNOS expression and NO production through activation of NF-κB and MAPK.


EPEC iNOS nitric oxide outer membrane protein NF-κB MAP kinase 


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  1. 1.
    Levine, M. M., and R. Edelman. 1984. Enteropathogenic Escherichia coli of classic serotypes associated with infant diarrhea: Epidemiology and pathogenesis. Epidemiol. Rev. 6:31–51.PubMedGoogle Scholar
  2. 2.
    Kenny, B., R. DeVinney, M. Stein, D. Reinscheid, E. Frey, and B. Finlay. 1997. Enteropathogenic E. coli (EPEC) transfers its receptor for intimate adherence into mammalian cells. Cell 91:511–520.CrossRefPubMedGoogle Scholar
  3. 3.
    Savkovic, S., A. Koutsouris, and G. Hecht. 1997. Activation of NF-κB in intestinal epithelial cells by enteropathogenic Escherichia coli. Am. J. Physiol. 273:C1160–C1167.PubMedGoogle Scholar
  4. 4.
    Savkovic, S., A. Koutsouris, and G. Hecht. 1996. Attachment of a noninvasive enteric pathogen, enteropathogenic Escherichia coli, to cultured human intestinal epithelial monolayers induces transmigration of neutrophils. Infect. Immun. 64:4480–4487.PubMedGoogle Scholar
  5. 5.
    Czerucka, D., S. Dahan, B. Mograbi, B. Rossi, and P. Rampal. 2001. Implication of mitogen-activated protein kinases in T84 cell responses to enteropathogenic Escherichia coli infection. Infect. Immun. 69:1298–1305.CrossRefPubMedGoogle Scholar
  6. 6.
    de Grado, M., C. M. Rosenberger, A. Gauthier, B. A. Vallance, and B. B. Finlay. 2001. Enteropathogenic Escherichia coli infection induces expression of the early growth response factor by activating mitogen-activated protein kinase cascades in epithelial cells. Infect. Immun. 69:6217–6224.CrossRefPubMedGoogle Scholar
  7. 7.
    Hecht, G. 2001. Microbes and microbial toxins: Paradigms for microbial mucosal interactions. VII. Enteropathogenic Escherichia coli: Physiological alterations from an extracellular position. Am. J. Physiol. Gastrointest. Liver Physiol. 281:G1–G7.PubMedGoogle Scholar
  8. 8.
    Savkovic, S. D., A. Ramaswamy, A. Koutsouris, and G. Hecht. 2001. EPEC activated ERK1/2 participate in inflammatory response but not tight junction barrier disruption. Am. J. Physiol. Gastrointest. Liver Physiol. 281:G890–G898.PubMedGoogle Scholar
  9. 9.
    Baldwin, T. J., S. F. Brooks, S. Knutton, H. A. Manjarrez Hernandez, A. Aitken, and P. H. Williams. 1990. Protein phosphorylation by protein kinase C in HEp-2 cells infected with enteropathogenic Escherichia coli. Infect. Immun. 58:761–765.PubMedGoogle Scholar
  10. 10.
    Rosenshine, I., M. Donnenberg, J. B. Kaper, and B. B. Finlay. 1992. 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. 11:3551–3560.PubMedGoogle Scholar
  11. 11.
    Celli, J., M. Olivier, and B. B. Finlay. 2001. Enteropathogenic Escherichia coli mediates antiphagocytosis through the inhibition of PI3-kinase-dependent pathways. EMBO J. 20:1245–1258.CrossRefPubMedGoogle Scholar
  12. 12.
    Witthoft, T., L. Eckmann, J. M. Kim, and M. F. Kagnoff. 1998. Enteroinvasive bacteria directly activate expression of iNOS and NO production in human colon epithelial cells. Am. J. Physiol. 275:G564–G571.PubMedGoogle Scholar
  13. 13.
    Vallance, B. A., W. Deng, M. D. Grado, C. Chan, K. Jacobson, and B. B. Finlay. 2002. Modulation of inducible nitric oxide synthase expression by attaching and effacing bacterial pathogen Citrobacter rodentium in infected mice. Infect. Immun. 70:6424–6435.CrossRefPubMedGoogle Scholar
  14. 14.
    Davis, R. J. 1993. The mitogen-activated protein kinase signal transduction pathway. J. Biol. Chem. 268:14553–14556.PubMedGoogle Scholar
  15. 15.
    Zhou, X., A. J. Giron, A. G. Torres, J. A. Crawford, E. Negrete, S. N. Vogel, and J. B. Kaper. 2003. Flagellin of enteropathogenic Escherichia coli stimulates interleukin-8 production in T84 cells. Infect. Immun. 71:2120–2129.CrossRefPubMedGoogle Scholar
  16. 16.
    Xie, Q.-W., Y. Kashiwabara, and C. Nathan. 1994. Role of transcription factor NF-κB/Rel in induction of nitric oxide synthase. J. Biol. Chem. 269:4705–4708.PubMedGoogle Scholar
  17. 17.
    Goldring, C. E. P., R. Narayanan, P. Lagadec, and J.-F. Jeannin. 1995. Transcriptional inhibition of the inducible nitric oxide synthase gene by competitive binding of NF-κB/Rel proteins. Biochem. Biophys. Res. Commun. 209:73–79.CrossRefPubMedGoogle Scholar
  18. 18.
    Kumar, S. S., V. Malladi, K. Sankaran, R. Haigh, P. H. Williams, and A. Balakrishnan. 2001. Extrusion of actin-positive strands from HEp-2 and Int 407 cells caused by outer membrane preparations of enteropathogenic Escherichia coil and specific attachment of wild type bacteria to the Strands. Can. J. Microbiol. 47:727–734.CrossRefPubMedGoogle Scholar
  19. 19.
    Kumar, S. S., K. Sankaran, R. Haigh, P. H. Williams, and A. Balakrishnan. 2001. Cytopathic effects of outer-membrane preparations of enteropathogenic Escherichia coli and co-expression of maltoporin with secretory virulence factor, EspB. J. Med. Microbiol. 50:602–612.PubMedGoogle Scholar
  20. 20.
    Malladi, V., B. Shankar, P. H. Williams, and A. Balakrishnan. 2004. Enteropathogenic Escherichia coli outer membrane proteins induce changes in cadherin junctions of caco-2 cells through activation of PKCα. Microbes Infect. 6:38–50.CrossRefPubMedGoogle Scholar
  21. 21.
    Finlay, B. B., I. Rosenshine, M. S. Donnenberg, and J. B. Kaper. 1992. Cytoskeletal composition of attaching and effacing lesions associated with enteropathogenic Escherichia coli adherence to HeLa cells. Infect. Immun. 60:2541–2543.PubMedGoogle Scholar
  22. 22.
    Knutton, S., T. Baldwin, P. H. Williams, and A. S. McNeish. 1989. 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. 57:1290–1298.PubMedGoogle Scholar
  23. 23.
    Baldini, M. M., J. B. Kaper, M. M. Levine, D. C. Candy, and H. W. Moon. 1983. Plasmid-mediated adhesion in enteropathogenic Escherichia coli. J. Pediatr. Gastroenterol. Nutr. 2:534–538.Google Scholar
  24. 24.
    Donnenberg, M. S., and J. B. Kaper. 1991. Construction of an eae deletion mutant of enteropathogenic Escherichia coli by using a positive-selection suicide vector. Infect. Immun. 59:4310–4317.Google Scholar
  25. 25.
    Jerse, A. E., K. G. Gicquelais, and J. B. Kaper. 1991. Plasmid and chromosomal elements involved in the pathogenesis of attaching and effacing Escherichia coli. Infect. Immun. 59:3869–3875.PubMedGoogle Scholar
  26. 26.
    Jerse, A. E., and J. B. Kaper. 1991. 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. 59:4302–4309.PubMedGoogle Scholar
  27. 27.
    Miyamoto, S., P. J. Chiao, and I. M. Verma. 1994. Enhanced IκBα degradation is responsible for constitutive NF-κB activity in mature murine B-cell lines. Mol. Cell. Biol. 14:3276–3282.PubMedGoogle Scholar
  28. 28.
    Narayanan, K., A. Balakrishnan, and S. Miyamoto. 2000. NF-κB is essential for induction of proinflammatory cytokine genes by filarial parasitic sheath proteins. Mol. Immunol. 37:115–123.CrossRefPubMedGoogle Scholar
  29. 29.
    Hall, L. R., R. K. Mehlotra, A. W. Higgins, M. A. Haxhiu, and E. Pearlman. 1998. An essential role for interleukin-5 and eosinophils in helminth-induced airway hyperresponsiveness. Infect. Immun. 66:4425–4430.PubMedGoogle Scholar
  30. 30.
    Green, L. C., D. A. Wagner, J. Glogowski, P. L. Skipper, J. S. Wishnok, and S. R. Tannenbaum. 1982. Analysis of nitrate, nitrite, and [15N] nitrate in biological fluids. Anal. Biochem. 126:131–138.CrossRefPubMedGoogle Scholar
  31. 31.
    Savkovic, S. D., A. Koutsouris, and G. Hecht. 2003. PKC ζ participates in activation of inflammatory response induced by enteropathogenic E. coli. Am. J. Physiol. Cell Physiol. 285:C512–C521.PubMedGoogle Scholar
  32. 32.
    Foubister, V., I. Rosenshine, and B. B. Finlay. 1994. A diarrheal pathogen, enteropathogenic Escherichia coli (EPEC), triggers a flux of inositol phosphates in infected epithelial cells. J. Exp. Med. 179:993–998.CrossRefPubMedGoogle Scholar
  33. 33.
    Kim, J. M., J. S. Kim, H. C. Jung, I. S. Song, and C. Y. Kim. 2002. Up-regulation of inducible nitric oxide synthase and nitric oxide in Helicobacter pylori-infected human gastric epithelial cells: Possible role of interferon-γ in polarized nitric oxide secretion. Helicobacter 7:116–125.CrossRefPubMedGoogle Scholar
  34. 34.
    Mysorekar, I. U., M. A. Mulvey, S. J. Hultgren, and J. I. Gordon. 2002. Molecular regulation of urothelial renewal and host defenses during infection with uropathogenic Escherichia coli. J. Biol. Chem. 277:7412–7419.CrossRefPubMedGoogle Scholar
  35. 35.
    Frankel, G., O. Lider, R. Hershkoviz, A. P. Mould, and S. G. Kachalsky. 1996. The cell-binding domain of intimin from enteropathogenic Escherichia coli binds to betal integrins. J. Biol. Chem. 271:20359–20364.CrossRefPubMedGoogle Scholar
  36. 36.
    Frankel, G., A. D. Phillips, L. R. Trabulsi, S. Knutton, G. Dougan, and S. Matthews. 2001. Intimin and the host cell—Is it bound to end in Tir(s)?. Trends. Microbiol. 9:214–218.CrossRefPubMedGoogle Scholar
  37. 37.
    Higgins, L. M., G. Frankel, I. Connerton, N. S. Goncalves, G. Dougan, and T. T. MacDonald. 1999. Role of bacterial intimin in colonic hyperplasia and inflammation. Science 285:588–591.CrossRefPubMedGoogle Scholar
  38. 38.
    Didierlaurent, A., J. C. Sirard, J. P. Kraehenbuhl, and M. R. Neutra. 2002. How the gut senses its content. Cell. Microbiol. 4:61–72.CrossRefPubMedGoogle Scholar
  39. 39.
    Giron, J. A., A. G. Torres, E. Freer, and J. B. Kaper. 2002. The flagella of enteropathogenic Escherichia coli mediate adherence to epithelial cells. Mol. Microbiol. 44:361–379.CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science + Business Media, Inc. 2004

Authors and Affiliations

  • Vasantha Malladi
    • 1
  • Manjula Puthenedam
    • 1
  • Peter H. Williams
    • 2
  • Arun Balakrishnan
    • 1
  1. 1.Centre for BiotechnologyAnna UniversityChennaiIndia
  2. 2.Department of Microbiology and ImmunologyUniversity of LeicesterUK

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