Production and translocation of Stx2 are stimulated by an hypervirulent E. coli O157:H7 in contact with human intestinal epithelial cell monolayers

MARIA M AMARAL; NICOLAS E GARIMANO; CRISTINA IBARRA

Florencia

Simposio; 10th VTEC International Symposium on Shiga Toxin (Verocytotoxin) Producing Escherichia coli Infections; 2018

IntroductionGI infection with Shiga toxin (Stx2)-producing enterohemorrhagicEscherichia coli causes bloody diarrhea, hemorrhagic colitisand hemolytic uremic syndrome (HUS). E. coliO157:H7 isthe most prevalent serotype associated with HUS and Stx2is its major virulence factor.However, mechanisms involved in the pathogenesis mediated by Stx2 and how toxins cross the intestinal epitheliumare largely unknown.Our aim was to study the effects of E. coliO157:H7 on human colonic epithelial cellsto better understand the means by which Stx2 induces diarrhea and translocate the intestinal barrier. Material and MethodsWe examined cell viability in human intestinal cell lines (HCT-8, Caco-2) after incubation with E. coliO157:H7 (O157:H7) compared tothe correspondingmutant lacking stx2 gene (O157:H7∆stx2)supplemented withStx2 at the same concentration as measured in the filter-sterilized supernatant from aO157:H7 culture grown to exponential phase.Cellswere grown in 96-well culture plate on growth arrested conditions and antibiotic free media. Cell viability was measuredby neutral red uptake after a 24 h total time frame, 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 of O157:H7∆stx2or its filtered culture supernatant (SN). Dextran-FITC was used as an indicator of paracellularpermeability and EDTA (0.1 mM)as a tight junction disruptor. Transepithelial electric resistance was monitored daily before and after treatments.Collected basal mediacytotoxicity was evaluated onVero cellsand Dextran-FITC was measured by fluorometry. ResultsThe cytotoxic effects induced by O157:H7 on HCT-8 and Caco-2 cell lines weresignificantly higher than those observed with Stx2 and O157:H7∆stx2 combined. Furthermore, maximum Stx2 traslocationwas observed after treatment with O157:H7∆stx2 compared to EDTA and bacterial SNtreatments. On the contrary, maximum Dextran-FITC passage was achieved with either EDTA or O157:H7∆stx2 treatment suggesting that O157:H7 could stimulate the Stx2 translocation across atranscellularin addition to paracellular pathway.These results indicate that a directcontact between O157:H7 and intestinal barrier could inducean increase on Stx2 production. Also, bacterial presence strongly enhances translocationacross the paracellular and transcellular pathways. Identification of host cell-derived factors and/orstructural cellular changes derived of bacteria-intestinal cells interaction responsible for increasing Stx2 production and translocationcould lead tonew strategies for modulating STEC infections. Further experiments adding specific translocation inhibitors could elucidate the mechanisms involved in the Stx2 transport across the intestinal barrier.