CONICET | Buscador de Institutos y Recursos Humanos

Introduction GIinfection with Shiga toxin (Stx2)-producing enterohemorrhagicEscherichia coli causes bloody diarrhea,hemorrhagic colitisand hemolytic uremic syndrome (HUS). E. coliO157:H7 isthe mostprevalent serotype associated with HUS and Stx2is its major virulence factor.However,mechanisms involved in the pathogenesis mediated by Stx2 and how toxins crossthe intestinal epitheliumare largely unknown.Our aimwas to study the effects of E. coliO157:H7on human colonic epithelial cellsto better understand the means by which Stx2induces diarrhea and translocate the intestinal barrier. Materialand MethodsWeexamined cell viability in human intestinal cell lines (HCT-8, Caco-2) after incubation with E. coliO157:H7 (O157:H7) comparedtothe correspondingmutant lacking stx2gene (O157:H7∆stx2)supplemented withStx2 at the same concentration as measuredin the filter-sterilized supernatant from aO157:H7 culture grown to exponentialphase.Cellswere grownin 96-well culture plate on growth arrested conditions and antibiotic freemedia. Cell viability was measuredby neutral red uptake after a 24 h total timeframe, having been exposed for 1 h or4 h to bacteria alone or with Stx2.We have also evaluated the translocation ofStx2(100ng/ml)across HCT-8 cells cultured as monolayers on Millicell insertsalone orin presence ofO157:H7∆stx2or its filtered culturesupernatant (SN). Dextran-FITC was used as an indicator of paracellularpermeability and EDTA (0.1 mM)as a tightjunction disruptor. Transepithelial electric resistance was monitored daily beforeand after treatments.Collectedbasal mediacytotoxicity was evaluated onVero cellsand Dextran-FITC was measuredby fluorometry. ResultsThe cytotoxiceffects induced by O157:H7 on HCT-8 and Caco-2 cell lines weresignificantlyhigher than those observed with Stx2 and O157:H7∆stx2 combined. Furthermore, maximumStx2 traslocationwas observed after treatment with O157:H7∆stx2 compared toEDTA and bacterial SNtreatments. On the contrary, maximum Dextran-FITC passagewas achieved with either EDTA or O157:H7∆stx2 treatment suggesting that O157:H7could stimulate the Stx2 translocation across atranscellularin addition toparacellular pathway.These results indicate that a directcontactbetween O157:H7 and intestinal barrier could inducean increase on Stx2production. Also, bacterial presence strongly enhances translocationacross theparacellular and transcellular pathways. Identification of host cell-derivedfactors and/orstructural cellular changes derived of bacteria-intestinal cells interactionresponsible for increasing Stx2 production and translocationcould lead tonew strategies formodulating STEC infections. Further experiments adding specific translocation inhibitorscould elucidate the mechanisms involved in the Stx2 transport across theintestinal barrier.