Análise celular e ultraestrutural dos mecanismos de patogênese de cepas de Escherichia coli enteroagregativa (EAEC) invasoras em células intestinais in vitro e ex vivo

Abstract

The diarrheal syndrome is considered a serious public health problem all over the world and is considered a major cause of morbidity and mortality in developing countries. The pathogenesis of enteroaggregative Escherichia coli (EAEC) remains uncertain and has been investigated extensively by the use of in vitro and, to a lesser extent, ex vivo models. The competence of EAEC to adhere to the intestinal epithelium of the host is a key role to the colonization and disease development. Further, EAEC can invade intestinal cells and cause severe alterations in the intestinal epithelium. The present study investigated the interaction of invasive EAEC strains isolated from childhood diarrhea with rabbit ileal and colonic mucosa ex vivo. EAEC strains were also evaluated for the ability of intracellular persistence associated with cytokine production in the T84 intestinal line. The viability of cells and occurrence of apoptosis/necrosis in in vitro T84 cell line was also evaluated. The adhesion assays were performed with the strains co-incubated with fragments of rabbit intestine over a period of 6 hours and were analysed by scanning electron microscopy (SEM). The intracellular persistence and production of cytokines by sandwich ELISA were evaluated upon 24 hours and the phase contrast and fluorescence microscopy and flow cytometry were conducted after 6, 16 and 24 hours of interaction with infected T84 cells. Through SEM we observed that all EAEC strains adhered to both intestinal sites (ileum and colon) with the typical aggregative adherence pattern of “stacked bricks”, however the highest degree of adherence was observed on colonic mucosa. The survival rates of EAEC H92/3, I49/3 and 042 were 1.4x103, 3.2x103 and 0.5x103 CFU/mL, respectively. All EAEC strains tested induced significant amounts of IL-8, IL-6 and TNF-α compared to uninfected T84 cells. Phase contrast microscopy showed loss of viability in cells infected for periods of 16 hours. By fluorescence microscopy, we observed that cells infected with EAEC H92/3 and I49/3 had nuclei damages. Furthermore, by flow cytometry, our data revealed that EAEC provoked late apoptosis and necrosis of T84 cells. These data showed that invasive EAEC strains may have a high tropism for the human colon. Invasive strains can survives within intestinal cell line, inducing a proinflammatory immune response and triggering apoptotic and/or necrotic processes in T84 cells. The different pathways of virulence investigated in the present study contribute to the understanding of the mechanism of pathogenicity of invasive EAEC.