CONICET | Buscador de Institutos y Recursos Humanos

Enterohaemorrhagic Escherichia coli O157:H7 (EHEC) strains are responsible for multiple clinical syndromes including bloody diarrhea, hemolytic uremic syndrome (HUS). HUS is a systemic diseasecaused mainly by Shiga toxin type 2 (Stx2). EHEC employs a type III secretion system to colonize the bowel and to inoculate effector proteins such as EspF. This protein is responsible to disrupt tight junctions, inhibit phagocytosis and induce effacement of microvilli and apoptosis. However, the role of EspF on the mechanisms related to Stx2 production across human intestine are not well known.The EHEC∆espF mutant was constructed by Chengsong Wan and co-workers (Zhao et al. PlosOne 2013). We have evaluated the equivalent amount of Stx2 on Vero cells (ATCC CCL-81) culturedwith the supernatants of EHEC wild type (SNwt) and EHEC ∆espF (SN∆espF).Both strains, EHEC O157:H7 wt and EHEC O157:H7 ∆espF were grown in LB medium for 18 h at 37 °C in LB with shaking at 150 rpm and then diluted 1:10 in DMEM/F12 medium with the addition of 10 mM of HEPES and grown to exponential phase (optical density at 630 nm of 0.3-0.4). Bacterial supernatants were collected after centrifugation at 10,000 g for 5 min and sterilized by filtration through a 0.22-µm-pore-size filter. The titers of Stx2 of filter-sterilized supernatants were determined on Vero cells.A significant cytotoxic effect was observed when monolayers of Vero cells were exposed to different concentrations of purified commercial Stx2 under growth-arrested conditions. The CD50 was maximal after 72 h of incubation. Both, SN wt and SN ∆espF showed similar degrees of cytotoxicity on Vero cells corresponding to equivalent amount of Stx2 calculated by a non-parametric Mann-Whitney test.This initial trial shows that the absence of the gene EspF might not significantly affect secretion of Stx2 in the bacterial culture. More studies are needed to evaluate the effect of EspF on Stx2 production of EHEC.