Efeitos das toxinas A e B do Clostridium difficile sobre a via de WNT/Beta-catenina em células epiteliais intestinais

Abstract

Clostridium difficile toxins A and B (TcdA and TcdB) are homologous glycosyltransferases that inhibit a group of small GTPases within host cells, but several mechanisms underlying their pathogenic activity remain unclear. Here, we evaluated the effects of TcdA and TcdB on the Wnt/Beta-catenin pathway, the major driving force behind the proliferation of epithelial cells in colonic crypts. IEC-6 and RKO cells stimulated with Wnt3a-conditioned medium were incubated with 10, 50 and 100 ng/mL of TcdA or TcdB for 24h, resulting in a dose-dependent inhibition of the Wnt signaling, as demonstrated by a T-cell factor (TCF) reporter assay. This was further confirmed by immunofluorescence staining for nuclear localization of Beta-catenin and western blotting for Beta-catenin and c-Myc (encoded by a Wnt target gene). Moreover, our western blot analysis showed a decrease in the Beta-catenin protein levels, which was reversed by z-VAD-fmk, a pan-caspase inhibitor. Nonetheless, TcdA was still able to inhibit the Wnt/Beta-catenin pathway even in the presence of z-VAD-fmk, lithium chloride (a GSK3B inhibitor), or constitutively active Beta-catenin, as determined by a TCF reporter assay. Furthermore, pre-incubation of RKO cells with TcdA for 12h also attenuated Wnt3a-mediated activation of Wnt signaling, suggesting that inactivation of Rho GTPases plays a significant role in that inhibition. Taken together, these findings suggest that attenuation of the Wnt signaling by TcdA and TcdB is important for their anti-proliferative effects.