E. coli O157:H7 stimulates Shiga toxin type 2 cytotoxic effects and translocation across human colonic epithelial cell lines

GARIMANO NICOLAS EZEQUIEL ; AMARAL MARÍA MARTA; CRISTINA IBARRA

Foz de Iguazú

Congreso; Congreso Brasilero de Microbiología; 2017

Sociedad Brasilera de Microbiología

Gastrointestinal infection with Shiga toxin (Stx2)-producing enterohemorrhagicEscherichia coli causes bloody diarrhea, hemorrhagic colitis and hemolyticuremic syndrome (HUS). E. coli O157:H7 is the most prevalent serotypeassociated with HUS and Stx2 is its major virulence factor. However, themechanisms involved in the pathogenesis of diarrhea mediated by Stx2 andhow toxins cross the intestinal epithelium are largely uncharacterized. Our aimwas to study the effects of E. coli O157:H7 on human colonic epithelial cells tobetter understand the means by which Stx2 induces diarrhea and translocatethe intestinal barrier. We examined cell viability in human intestinal cell lines(HCT-8, Caco-2) after incubation with purified Stx2, E. coli O157:H7 strain125/99 (O157:H7), its mutant lacking stx2 gene (O157:H7∆stx2) and filteredO157:H7 supernatant. Cells were grown in 96-well culture plate and viabilitywas measured by neutral red uptake after 1, 4 or 24h incubation period undergrowth-arrested condition. We have also evaluated the translocation of Stx2across HCT-8 cells cultured as monolayers on Millicell inserts in presence ofO157:H7∆stx2 or O157:H7∆stx2-SN. Dextran-FITC was used as an indicator ofparacellular permeability and EDTA (1 mM) as a disruptor of tight junctions.Transepithelial electric resistance was monitored daily during the developmentof cell culture, and after treatments. Collected basal media cytotoxicity wasevaluated on Vero cells and Dextran-FITC was measured by fluorometry. Aconcomitant increase of both parameters was observed in the presence ofO157:H7∆stx2 or its corresponding O157:H7∆stx2-SN on the luminal side.Furthermore, the cytotoxic effect induced by O157:H7 on HCT-8 and Caco-2 cell lines was significantly higher than those observed with Stx2 andO157:H7∆stx2 combined. These results indicate that both transcellular andparacellular pathways are implicated on Stx2 translocation, and that directcontact between O157:H7 and intestinal barrier induce an elevated cytotoxicresponse that suggest an increase on Stx2 production.