Stx2 translocation pathways across human intestinal epithelial cells are differentially stimulated by an hypervirulent E. coli O157:H7

GARIMANO NICOLÁS; IBARRA CRISTINA; AMARAL MARÍA MARTA

Mar del Plata

Congreso; LXIII Reunión Anual de la Sociedad Argentina de Investigación Clínica, LXVI Reunión Annual de la Sociedad Argentina de Inmunología, Reunión Anual de la Sociedad Argentina de Fisiología.; 2018

Sociedad Argentina de Investigación Clínica, Sociedad Argentina de Fisiología, Sociedad de Inmunología

Gastrointestinal infection with Shiga toxin (Stx2)-producing Escherichia coli causes bloody diarrhea, hemorrhagic colitis and hemolytic uremic syndrome (HUS). E. coli O157:H7 is the most prevalent serotype associated with HUS and Stx2 is its major virulence factor. However, mechanisms involved in pathogenesis mediated by Stx2 and how toxins cross the intestinal epithelium are unknown. Our aim was to study the effects of E. coli O157:H7 on human colonic epithelial cells to better understand the means by which Stx2 induces diarrhea and translocate the intestinal barrier. We have evaluated translocation of Stx2 across HCT-8 cells cultured as monolayers on Millicell inserts in presence of a O157:H7 mutant lacking stx2 gene (O157:H7∆stx2) or its filtered culture supernatant (SN O157:H7∆stx2). Additionally, O157:H7∆stx2 effects were evaluated after a 30 min pre-incubation with pathway-specific endocytic inhibitors: Amiloride (1mM), Dynasore (80 µM) and Methyl-β-CycloDextrin (MβCD, 4mM). Transepithelial electric resistance was monitored before and after treatments. Dextran-FITC was used as an indicator of paracellular pathway and EDTA (0.1 mM) as a tight junction disruptor. Collected basal media cytotoxicity was evaluated on Vero cells and Dextran-FITC was measured by fluorometry. Maximum Stx2 translocation was observed after treatment with O157:H7∆stx2 supplemented with Stx2 compared to EDTA and, lastly, bacterial SN treatment. Moreover, maximum Dextran-FITC passage was achieved with EDTA. MβCD showed the highest Stx2 translocation inhibition, followed by significant inhibition by Amiloride and Dynasore (p