Resumo

Atypical Enteropathogenic Escherichia coli (aEPEC) are frequent diarrheal agents worldwide. We previously showed that aEPEC 1551-2 invades intestinal T84 cells preferentially through the basolateral surface. To reach this surface, Salmonella, Shigella and Yersinia cross the intestinal epithelium through M cells of the follicle associated epithelium covering Peyer’s patches (PP), and promote antigen transcytosis as part of mucosal immune response. In this work, we evaluated the contribution of the Type Three Secretion System (T3SS) to aEPEC 1551-2 ability to: invade and persist within intestinal Caco-2 cells, translocate through M-like cells, colonize rat intestine, and translocate from the intestinal lumen to organs. An isogenic T3SS mutant was constructed by insertion mutagenesis of the escN gene. A recombinant plasmid pEscN was used in order to complement the escN mutant strain. Caco-2 cells (10⁵) were cultured for 10 d to reach differentiation. Invasiveness was evaluated after infection (10⁷ CFU/well) for 6 h, following gentamicin treatment, cell lysis and CFU counting. To obtain M-like cells, Caco-2 cells (10⁵) were cultured on the upper chamber of filter membranes till they reached a transmembrane electric resistance of ~420 Ω cm²; Raji-B cells (10⁶) were then added to the lower chamber and kept on culture for 6 d. Either Caco-2 or M-like cells were infected (10⁷ CFU/filter) with aEPEC 1551-2 or mutant strains and incubated for 6 h. Medium from the lower chamber was collected and CFU were quantified. The ability to colonize rat small intestine was tested in ileum fragments containing PP from female Wistar-EPM rats after 2 h of infection (10⁷ CFU/1 cm²), following transmission electron microcopy analysis. Bacterial translocation was induced in the same rat lineage by oroduodenal catheterization and bacterial inoculation (10 mL of 10¹⁰ CFU/mL suspension, confined between the duodenum and ileum with ligatures). After 2 h, mesenteric lymph nodes, liver and spleen were cultured for bacterial recovery. Results showed that aEPEC 1551-2 may: invade and persist within Caco-2 cells up to 24 h; translocate through M-like cells; promote characteristic attaching-effacing lesions on intestinal mucosa, and translocate to mesenteric lymph nodes and liver. The T3SS mutant lacked these properties. Altogether, these findings suggest that the T3SS of aEPEC 1551-2 is important to its ability to invade enterocytes and reach extraintestinal sites by M cells transcytosis.