Resumo

Atypical EnteropathogenicEscherichia coli(aEPEC) inject various effector proteins into intestinal cells through a Type Three Secretion System (T3SS) promoting attaching effacing lesions (A/E). We investigated the role of T3SS in the ability of aEPEC 1711-4 strain to associate with eukaryotic cells vitroand to translocate in anin vivo (BT). A mutant deficient in T3SS was obtained by homologous recombination (Lambda Red system) for escN gene deletion. Caco-2 cells were infected with the wild-type strain (1711-4WT), an isogenic mutant deficient in escN (1711-4ΔescN) or 1711-4ΔescN complemented with cloned escN 1711-4ΔescN (pEscN). The number of cell-associated bacteria 3 h after infection and those with ability to invade and/or persist intracellularly for 3 and 48 h, were quantified after eukaryotic cell lysis, before and after treatment with gentamicin. For BT experiments aEPEC 1711-4WT, 1711-4ΔescN or controls were inoculated in EPM-Wistar rats (n=11), by oroduodenal gavage and subsequent ligation of the duodenum and terminal ileum. After 2 h, mesenteric lymph nodes (MLN), spleen and liver, were harvested and cultured to determine the ratio number of bacteria/tissue mass (CFU/g). The escN mutant lost the ability to promote A/E lesion while the complemented strain was capable of producing A/E as observed by the fluorescent actin staining test in vitro. Concerning bacterial association in vitro, when testing the escN mutant (4x104 CFU), there was a marked reduction when compared to 1711-4WT (1.2x10E7 CFU) or the complemented mutant (1x10E7 CFU) (p <0.05). The escN mutant (4x10E5 CFU) also had a marked reduction in the ability to invade Caco-2 cells when compared to 1711-4 WT (3x10E3 CFU), while the complemented mutant had this ability restored (p <0.05). Although all strains persisted in the intracellular compartment for at least 48 h, the number of bacteria recovered when testing the escNmutant was fivefold less than that observed with 1711-4WT (3x10E4 CFU) (p <0.05). Concerning translocation in vivo, the number of bacteria recovered from MLN, liver and spleen was higher for 1711-4WT when compared with the mutant deficient in T3SS (1711-4ΔescN) (p <0.05). This mutant was not recovered from the liver or spleen. These findings indicate that T3SS secreted effector proteins are required for efficient association of aEPEC 1711-4 in vivoas well for bacterial tranlocation in animal model.