Effects of E. coli O157:H7 on human colonic epitelial cell lines and translocation of Shiga toxin.

GARIMANO NICOLAS; ALBANESE ADRIANA; AMARAL MARÍA MARTA; IBARRA CRISTINA

Mar del Plata

Congreso; LXI Reunión Anual de la Sociedad Argentina de Investigación Clínica, LXIV Reunión Anual de la Sociedad Argentina de Inmunología y XLVIII Reunión Anual de la Sociedad Argentina de Farmacología Experimental.; 2016

Sociedad Argentina de Investigación Clínica

Shiga toxin (Stx)-producing Escherichia coli (STEC) strainsare responsible of bloody diarrhea (hemorrhagic colitis) and he-molytic uremic syndrome (HUS). STEC O157:H7 is, by far, the most prevalent serotype associated with HUS and Stx2 is themajor virulence factor associated for the more severe symptomsof the infection. After passage through the acidic barrier, STECcolonizes the human colon promoting production and absorptionof Stx2. However, the mechanisms involved in the pathogenesisof diarrhea mediated by Stx2 are not well known yet. Our aim wasto study the cytotoxic effects of STEC O157:H7 on human colonicepithelial cells in order to better understand the means by whichStx2 induces diarrhea and translocate the intestinal barrier. In thisstudy, we examined HCT-8 and Caco-2 viability after incubationwith purified Stx2, STEC O157:H7 strain 125/99 (125/99wt), amutant of 125/99 strain lacking stx2 gene (125/99Δstx2) and thefiltered 125/99wt supernatants. Cells were grown in 96-well cultureplate and viability was measured by neutral red uptake after a 24hincubation period under growth arrested condition. We have alsoevaluated the translocation of purified Stx2 across HCT-8 culturedas monolayers on Millicell cell culture inserts in the presence of125/99Δstx2. Transepithelial electric resistance was monitoreddaily during the development of cell culture until confluence wasachieved, and after Stx2 treatment to ensure monolayer integrity.Cytotoxicity of collected basal media on Vero cells assays wasenhanced by bacterial presence on the luminal side. Furthermore,the cytotoxic effects induced by 125/99wt and 125/99Δstx2 strainson HCT-8 and Caco-2 cell lines were significantly higher than thoseobserved with purified Stx2 and filtered 125/99wt supernatants.These results indicate the importance of bacterial cells in theinteraction and translocation of Stx2 through the intestinal barrierto cause HUS.