A transcelullar Gb3 dependet pathway is mainly responsible for Shiga toxins-2 cytotoxicity and translocation across human intestinal epithelial cells infected with E. coli O157:H7.

AMARAL MARÍA MARTA; GARIMANO NICOLÁS; IBARRA CRISTINA

Mar del Plata

Congreso; LXIV Reunión Anual de la Sociedad Argentina de Investigación Clínica.; 2019

Sociedad Argentina de Investigación Clínica

Shiga toxin-2 (Stx2) is produced andreleased by E. coli O157:H7 (O157:H7) into the intestinal lumenafter colonization, and is able to translocate to the circulatorysystem and reach target cells causing hemolytic uremicsyndrome. Our aim was to elucidate which pathways wereinvolved in Stx2 endocytosis and translocation across intestinalcells infected with STEC. HCT-8 cells grown on 96-well plateswere preincubated with specific endocytosis inhibitors such asEliglustat (EG), Dynasore (DY), MßCD or Amiloride (AM). Then,cells were washed and incubated for 4 h with 100 ng/ml Stx2alone or in the presence of O157:H7 mutant lacking stx2 gene(O157:H7Ðstx2). Stx2 uptake was measured by flow cytometryand its cytotoxic effect by neutral red uptake assay.Translocation of Stx2 was evaluated by inhibitor preincubation ofHCT-8, grown as monolayers on Millicell inserts, and incubatedwith O157:H7Ðstx2+ Stx2. Then Stx2 cytotoxicity was quantifiedin lower chamber media by neutral red uptake, To analyzeinhibitors effect on bacteria attachment, bacterial adherenceassays were performed on HCT-8 monolayers cultured on 24-wells plates. EG caused the maximum decrease of Stx2 cytotoxicactivity, followed by MßCD. AM and DY significantly neutralizedStx2 cytotoxicity but only in presence of O157:H7Ðstx2.Furthermore, Stx2 uptake was reduced when cells were preincubated with EG or MßCD, compared to DY or AM (p