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

GARIMANO NICOLAS EZEQUIEL; AMARAL MARÍA MARTA; IBARRA CRISTINA

Mar del Plata

Congreso; Reunión Conjunta SAIC SAI SAFIS 2018; 2018

SAIC-SAI-SAFIS

Gastrointestinal infection with Shiga toxin (Stx2)-producing Escherichia coli causes bloody diarrhea, hemorrhagic colitis and hemolyticuremic 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 Stx2and how toxins cross the intestinal epithelium are unknown. Our aimwas to study the effects of E. coli O157:H7 on human colonic epithelial cells to better understand the means by which Stx2 inducesdiarrhea and translocate the intestinal barrier.We have evaluated translocation of Stx2 across HCT-8 cells culturedas monolayers on Millicell inserts in presence of a O157:H7 mutantlacking stx2 gene (O157:H7∆stx2) or its filtered culture supernatant(SNO157:H7∆stx2). Additionally, O157:H7∆stx2 effects were evaluated after a 30 min pre-incubation with pathway-specific endocyticinhibitors: 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 anindicator of paracellular translocation and EDTA (0.1 mM) as a tightjunction disruptor. Collected basal media cytotoxicity was evaluatedon Vero cells and Dextran-FITC was measured by fluorometry.Maximum Stx2 translocation was observed after treatment withO157:H7∆stx2 supplemented with Stx2 compared to EDTA and,lastly, bacterial SN treatment. Additionally, maximum Dextran-FITCpassage was achieved with EDTA. MβCD showed the highest Stx2translocation inhibition, followed by significant inhibition by Amilorideand Dynasore (p