|362-1||The influence of environmental factors on the adherence pattern and interaction of a strain of atypical enteropathogenic Escherichia coli|
|Autores:||Fabiano Teodoro Romão (UNIFESP - Universidade Federal de São Paulo) ; Rodrigo Tavanelli Hernandes (UNESP - Universidade Estadual de São Paulo) ; Denise Yamamoto (UNIFESP - Universidade Federal de São Paulo) ; Tânia Aparecida Tardelli Gomes do Amaral (UNIFESP - Universidade Federal de São Paulo) |
Enteropathogenic Escherichia coli (EPEC) are agents of human diarrhea, which produce attaching-effacing lesions (AE) that are characterized by effacement of microvilli and intimate bacterial adherence to enterocytes. The AE lesion occurs after the interaction between intimin (a bacterial outer membrane protein) and its receptor, Tir, which is translocated into eukaryotic cells by a type 3 secretion system. EPEC are divided into typical (tEPEC) and atypical (aEPEC). tEPEC produces the Bundle-forming pilus (BFP), which mediates the localized adherence (LA) pattern that is characterized by formation of compact bacterial microcolonies on cell surfaces. Although aEPEC are devoid of BFP and thus they produce loose microcolonies, we had previously identified some aEPEC strains producing compact LA. However, occasionally, we noted that alterations in pH of culture medium modified the original adherence pattern but the factors that change the expression of the genes involved in this interaction, as an adaption to environmental variations, remain unclear. In this study, we evaluated the influence of pH, glucose and CO2 concentration, and presence of bile salts in the adhesiveness of aEPEC 1551-2, by determining the number of cell associated-bacteria and by analyzing the maintenance of the LA pattern on HeLa cells grown in Dulbecco Modified Eagle Medium (DMEM) with 2% fetal calf serum. Bacterial suspensions grown in Luria Bertani broth (18 h, 37°C) (~10e8 bacteria/well) were inoculated in incomplete cell monolayers in DMEM with different pHs (6.0; 7.2 or 8.0), glucose (5 mM or 25 mM) or CO2 concentrations (0.03% and 5%), and bile salts (0% or 0.015% w/v). After 6 h of incubation, cells were lysed (1% Triton X-100), supernatants were diluted and seeded in triplicate, and the CFU were counted (3 assays in triplicated). The results showed that the efficiency of aEPEC 1551-2 adherence to HeLa cells is higher between pH 7.2 and 8.0, and bacterial growth rate was unaffected under these different pHs. Moreover, as glucose concentration raised, aEPEC 1551-2 tended to adhere preferentially to abiotic surfaces; this feature is probably associated with type I fimbriae (T1P) production, since an isogenic T1P mutant no longer adhered to the abiotic surfaces, even in high glucose concentrations. Higher CO2 concentrations propitiated the most efficient bacterial interaction to HeLa cells. The LA pattern was maintained only at pH7.2 in DMEM with low glucose independently of the CO2 concentration. The presence of bile salts promoted no alterations in bacterial interaction at the concentrations used. Considering the different environmental features of the human intestines, our data may suggest that aEPEC 1551-2 can associate similarly to different parts of this organ.
Palavras-chave: aEPEC, Regulation, Environmental Factors, Adherence Pattern, Interaction