Article | Published:

EPEC NleH1 is significantly more effective in reversing colitis and reducing mortality than NleH2 via differential effects on host signaling pathways

Laboratory Investigationvolume 98pages477488 (2018) | Download Citation


Enteropathogenic Escherichia coli (EPEC) is a foodborne pathogen that uses a type III secretion system to translocate effector molecules into host intestinal epithelial cells (IECs) subverting several host cell processes and signaling cascades. Interestingly, EPEC infection induces only modest intestinal inflammation in the host. The homologous EPEC effector proteins, NleH1 and NleH2, suppress the nuclear factor-κB (NF-κB) pathway and apoptosis in vitro. Increased apoptosis and activation of NF-κB and MAP kinases (MAPK) contribute to the pathogenesis of inflammatory bowel diseases (IBD). The aim of this study was to determine if NleH1 and NleH2 also block MAPK pathways in vitro and in vivo, and to compare the effects of these bacterial proteins on a murine model of colitis. Cultured IECs were infected with various strains of EPEC expressing NleH1 and NleH2, or not, and the activation of ERK1/2 and p38 was determined. In addition, the impact of infection with various strains of EPEC on murine DSS colitis was assessed by change in body weight, colon length, histology, and survival. Activation of apoptosis and MAPK signaling were also evaluated. Our data show that NleH1, but not NleH2, suppresses ERK1/2 and p38 activation in vitro. Interestingly, NleH1 affords significantly greater protection against and hastens recovery from dextran sodium sulfate (DSS)-induced colitis compared to NleH2. Strikingly, colitis-associated mortality was abolished by infection with EPEC strains expressing NleH1. Interestingly, in vivo NleH1 suppresses activation of ERK1/2 and p38 and blocks apoptosis independent of the kinase domain that inhibits NF-κB. In contrast, NleH2 suppresses only caspase-3 and p38, but not ERK1/2. We conclude that NleH1 affords greater protection against and improves recovery from DSS colitis compared to NleH2 due to its ability to suppress ERK1/2 in addition to NF-κB, p38, and apoptosis. These findings warrant further investigation of anti-inflammatory bacterial proteins as novel treatments for IBD.

  • Subscribe to Laboratory Investigation for full access:



Additional access options:

Already a subscriber?  Log in  now or  Register  for online access.


  1. 1.

    Dean P, Kenny B. The effector repertoire of enteropathogenic E. coli: ganging up on the host cell. Curr Opin Microbiol. 2009;12:101–9.

  2. 2.

    Akira S, Uematsu S, Takeuchi O. Pathogen recognition and innate immunity. Cell. 2006;124:783–801.

  3. 3.

    Morrison DK. MAP kinase pathways. Cold Spring Harb Perspect Biol. 2012;4:a011254

  4. 4.

    Gaestel M, Kotlyarov A, Kracht M. Targeting innate immunity protein kinase signalling in inflammation. Nat Rev Drug Discov. 2009;8:480–99.

  5. 5.

    Guma M, Stepniak D, Shaked H, et al. Constitutive intestinal NF-kappaB does not trigger destructive inflammation unless accompanied by MAPK activation. J Exp Med. 2011;208:1889–1900.

  6. 6.

    Quetglas EG, Mujagic Z, Wigge S, et al. Update on pathogenesis and predictors of response of therapeutic strategies used in inflammatory bowel disease. World J Gastroenterol. 2015;21:12519–43.

  7. 7.

    Broom OJ, Widjaya B, Troelsen J, et al. Mitogen activated protein kinases: a role in inflammatory bowel disease? Clin Exp Immunol. 2009;158:272–80.

  8. 8.

    Ihara E, Beck PL, Chappellaz M, et al. Mitogen-activated protein kinase pathways contribute to hypercontractility and increased Ca2+ sensitization in murine experimental colitis. Mol Pharmacol. 2009;75:1031–41.

  9. 9.

    Li YY, Yuece B, Cao HM, et al. Inhibition of p38/Mk2 signaling pathway improves the anti-inflammatory effect of WIN55 on mouse experimental colitis. Lab Invest. 2013;93:322–33.

  10. 10.

    Kwon KH, Ohigashi H, Murakami A. Dextran sulfate sodium enhances interleukin-1 beta release via activation of p38 MAPK and ERK1/2 pathways in murine peritoneal macrophages. Life Sci. 2007;81:362–71.

  11. 11.

    Samak G, Chaudhry KK, Gangwar R, et al. Calcium/Ask1/MKK7/JNK2/c-Src signalling cascade mediates disruption of intestinal epithelial tight junctions by dextran sulfate sodium. Biochem J. 2015;465:503–15.

  12. 12.

    Miampamba M, Larson G, Lai C, et al. RDEA110, a potent and highly selective MEK1/2 inhibitor is beneficial in dextran sulfate sodium (DSS)-induced chronic colits in mice. The ACG annual scientific meeting and postgraduate course, Orlando, FL, 3–8 October 2008.

  13. 13.

    Marrero JA, Matkowskyj KA, Yung K. et al. Dextran sulfate sodium-induced murine colitis activates NF-kappaB and increases galanin-1 receptor expression. Am J Physiol Gastrointest Liver Physiol. 2000;278:G797–804.

  14. 14.

    Alex P, Zachos NC, Nguyen T, et al. Distinct cytokine patterns identified from multiplex profiles of murine DSS and TNBS-induced colitis. Inflamm Bowel Dis. 2009;15:341–52.

  15. 15.

    Perse M, Cerar A. Dextran sodium sulphate colitis mouse model: traps and tricks. J Biomed Biotechnol. 2012;2012:718617.

  16. 16.

    Sharma R, Tesfay S, Tomson FL, et al. Balance of bacterial pro- and anti-inflammatory mediators dictates net effect of enteropathogenic Escherichia coli on intestinal epithelial cells. Am J Physiol Gastrointest Liver Physiol. 2006;290:G685–94.

  17. 17.

    Reis RS, Horn F. Enteropathogenic Escherichia coli, Samonella, Shigella and Yersinia: cellular aspects of host-bacteria interactions in enteric diseases. Gut Pathog. 2010;2:8. -4749-2-8

  18. 18.

    Wong AR, Pearson JS, Bright MD, et al. Enteropathogenic and enterohaemorrhagic Escherichia coli: even more subversive elements. Mol Microbiol. 2011;80:1420–38.

  19. 19.

    Hemrajani C, Berger CN, Robinson KS, et al. NleH effectors interact with Bax inhibitor-1 to block apoptosis during enteropathogenic Escherichia coli infection. Proc Natl Acad Sci USA. 2010;107:3129–34.

  20. 20.

    Royan SV, Jones RM, Koutsouris A, et al. Enteropathogenic E. coli non-LEE encoded effectors NleH1 and NleH2 attenuate NF-kappaB activation. Mol Microbiol. 2010;78:1232–45.

  21. 21.

    Gao X, Wan F, Mateo K, et al. Bacterial effector binding to ribosomal protein s3 subverts NF-kappaB function. PLoS Pathog. 2009;5:e1000708.

  22. 22.

    Pham TH, Gao X, Tsai K, et al. Functional differences and interactions between the Escherichia coli type III secretion system effectors NleH1 and NleH2. Infect Immun. 2012;80:2133–40.

  23. 23.

    Grishin AM, Condos TE, Barber KR, et al. Structural basis for the inhibition of host protein ubiquitination by Shigella effector kinase OspG. Structure. 2014;22:878–88.

  24. 24.

    Halavaty AS, Anderson SM, Wawrzak Z, et al. Type III effector NleH2 from Escherichia coli O157:H7 str. Sakai features an atypical protein kinase domain. Biochemistry. 2014;53:2433–5.

  25. 25.

    de Jong MF, Liu Z, Chen D, et al. Shigella flexneri suppresses NF-kappaB activation by inhibiting linear ubiquitin chain ligation. Nat Microbiol. 2016;1:16084.

  26. 26.

    Zhou H, Monack DM, Kayagaki N, et al. Yersinia virulence factor YopJ acts as a deubiquitinase to inhibit NF-kappa B activation. J Exp Med. 2005;202:1327–32.

  27. 27.

    Johannessen M, Askarian F, Sangvik M, et al. Bacterial interference with canonical NFkappaB signalling. Microbiology. 2013;159:2001–13.

  28. 28.

    Wu H, Jones RM, Neish AS. The Salmonella effector AvrA mediates bacterial intracellular survival during infection in vivo. Cell Microbiol. 2012;14:28–39.

  29. 29.

    Rhee KJ, Cheng H, Harris A, et al. Determination of spatial and temporal colonization of enteropathogenic E. coli and enterohemorrhagic E. coli in mice using bioluminescent in vivo imaging. Gut Microbes. 2011;2:34–41.

  30. 30.

    Savkovic SD, Koutsouris A, Hecht G. Attachment of a noninvasive enteric pathogen, enteropathogenic Escherichia coli, to cultured human intestinal epithelial monolayers induces transmigration of neutrophils. Infect Immun. 1996;64:4480–7.

  31. 31.

    Baruch K, Gur-Arie L, Nadler C, et al. Metalloprotease type III effectors that specifically cleave JNK and NF-kappaB. EMBO J. 2011;30:221–31.

  32. 32.

    Sham HP, Shames SR, Croxen MA, et al. Attaching and effacing bacterial effector NleC suppresses epithelial inflammatory responses by inhibiting NF-kappaB and p38 mitogen-activated protein kinase activation. Infect Immun. 2011;79:3552–62.

  33. 33.

    Ruchaud-Sparagano MH, Maresca M, Kenny B. Enteropathogenic Escherichia coli (EPEC) inactivate innate immune responses prior to compromising epithelial barrier function. Cell Microbiol. 2007;9:1909–21.

  34. 34.

    Nguyen M, Rizvi J, Hecht G. Expression of enteropathogenic Escherichia coli map is significantly different than that of other type III secreted effectors in vivo. Infect Immun. 2015;83:130–7.

  35. 35.

    Zhuang X, Chen Z, He C, et al. Modulation of host signaling in the inflammatory response by enteropathogenic Escherichia coli virulence proteins. Cell Mol Immunol. 2017;14:237–44.

  36. 36.

    Grishin AM, Cherney M, Anderson DH, et al. NleH defines a new family of bacterial effector kinases. Structure. 2014;22:250–9.

  37. 37.

    de Jong MF, Alto NM. Thinking outside the Osp(G)--kinase activation by E2-ubiquitin. EMBO J. 2014;33:403–4.

  38. 38.

    Grishin AM, Beyrakhova KA, Cygler M. Structural insight into effector proteins of Gram-negative bacterial pathogens that modulate the phosphoproteome of their host. Protein Sci. 2015;24:604–20.

  39. 39.

    Blasche S, Arens S, Ceol A, et al. The EHEC-host interactome reveals novel targets for the translocated intimin receptor. Sci Rep. 2014;4:7531.

  40. 40.

    Pham TH, Gao X, Singh G, et al. Escherichia coli virulence protein NleH1 interaction with the v-Crk sarcoma virus CT10 oncogene-like protein (CRKL) governs NleH1 inhibition of the ribosomal protein S3 (RPS3)/nuclear factor kappaB (NF-kappaB) pathway. J Biol Chem. 2013;288:34567–74.

  41. 41.

    Feuerbacher LA, Hardwidge PR. Influence of NleH effector expression, host genetics, and inflammation on Citrobacter rodentium colonization of mice. Microbes Infect. 2014;16:429–33.

  42. 42.

    te Velde AA, de Kort F, Sterrenburg E, et al. Comparative analysis of colonic gene expression of three experimental colitis models mimicking inflammatory bowel disease. Inflamm Bowel Dis. 2007;13:325–30.

  43. 43.

    Coskun M, Olsen J, Seidelin JB, et al. MAP kinases in inflammatory bowel disease. Clin Chim Acta. 2011;412:513–20.

  44. 44.

    Ye F, Zhang M. Structures and target recognition modes of PDZ domains: recurring themes and emerging pictures. Biochem J. 2013;455:1–14.

  45. 45.

    Donowitz M, Cha B, Zachos NC, et al. NHERF family and NHE3 regulation. J Physiol. 2005;567:3–11.

  46. 46.

    Martinez E, Schroeder GN, Berger CN, et al. Binding to Na(+) /H(+) exchanger regulatory factor 2 (NHERF2) affects trafficking and function of the enteropathogenic Escherichia coli type III secretion system effectors Map, EspI and NleH. Cell Microbiol. 2010;12:1718–31.

  47. 47.

    Ardura JA, Friedman PA. Regulation of G protein-coupled receptor function by Na+/H+ exchange regulatory factors. Pharmacol Rev. 2011;63:882–900.

  48. 48.

    Birge RB, Kalodimos C, Inagaki F, et al. Crk and CrkL adaptor proteins: networks for physiological and pathological signaling. Cell Commun Signal. 2009;7:13. -811X-7-13

  49. 49.

    Suzuki M, Mimuro H, Suzuki T, et al. Interaction of CagA with Crk plays an important role in Helicobacter pylori-induced loss of gastric epithelial cell adhesion. J Exp Med. 2005;202:1235–47.

Download references


We thank members of Hecht lab for providing invaluable comments. This research was supported by National Institutes of Health grant (DK097043 to G.H.) and the Department of Veterans Affairs (BX000785 and BX002687 to G.H.).

Author information


  1. Department of Medicine, Division of Gastroenterology and Nutrition, Loyola University Chicago, Maywood, IL, USA

    • Sarah E. Kralicek
    • , Rocio Tapia
    •  & Gail Hecht
  2. Cortexyme Inc., South San Francisco, CA, USA

    • Mai Nguyen
  3. Department of Biomedical Laboratory Science, College of Health Sciences, Yonsei University at Wonju, Wonju, Gangwon-do, Republic of Korea

    • Ki-Jong Rhee
  4. Department of Microbiology and Immunology, Loyola University Chicago, Maywood, IL, USA

    • Gail Hecht
  5. Edward Hines Jr. VA Hospital, Hines, IL, USA

    • Gail Hecht


  1. Search for Sarah E. Kralicek in:

  2. Search for Mai Nguyen in:

  3. Search for Ki-Jong Rhee in:

  4. Search for Rocio Tapia in:

  5. Search for Gail Hecht in:

Conflict of interest

The authors declare that they have no conflict of interest.

Corresponding author

Correspondence to Gail Hecht.

Electronic supplementary material

About this article

Publication history






Rights and permissions

To obtain permission to re-use content from this article visit RightsLink.