Departamento de Fisiologia & Farmacologia, Faculdade de Medicina, Universidade Federal do Ceará (UFC), Fortaleza, Brazil.
Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro (UFRJ), Rio de Janeiro, Brazil.
Infect Immun. 2014 Jul;82(7):2680-7. doi: 10.1128/IAI.00567-13. Epub 2014 Apr 7.
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. In this study, we evaluated the effects of TcdA on the Wnt/β-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 for 24 h, 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 β-catenin and Western blotting for β-catenin and c-Myc (encoded by a Wnt target gene). Moreover, our Western blot analysis showed a decrease in the β-catenin protein levels, which was reversed by z-VAD-fmk, a pan-caspase inhibitor. Nonetheless, TcdA was still able to inhibit the Wnt/β-catenin pathway even in the presence of z-VAD-fmk, lithium chloride (a GSK3β inhibitor), or constitutively active β-catenin, as determined by a TCF reporter assay. Furthermore, preincubation of RKO cells with TcdA for 12 h 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 is important for TcdA antiproliferative effects.
艰难梭菌毒素 A 和 B（TcdA 和 TcdB）是同源糖基转移酶，可抑制宿主细胞内的一组小 GTP 酶，但它们致病活性的几个机制仍不清楚。在这项研究中，我们评估了 TcdA 对 Wnt/β-连环蛋白途径的影响，该途径是结肠隐窝上皮细胞增殖的主要驱动力。用 Wnt3a 条件培养基刺激 IEC-6 和 RKO 细胞后，将其与 10、50 和 100 ng/ml 的 TcdA 孵育 24 小时，结果显示 Wnt 信号呈剂量依赖性抑制，这通过 T 细胞因子（TCF）报告基因检测得到证实。这通过核定位 β-连环蛋白的免疫荧光染色和 β-连环蛋白和 c-Myc（Wnt 靶基因编码）的 Western blot 进一步得到证实。此外，我们的 Western blot 分析显示 β-连环蛋白蛋白水平下降，这被 pan-caspase 抑制剂 z-VAD-fmk 逆转。尽管如此，TcdA 仍能够抑制 Wnt/β-连环蛋白途径，即使存在 z-VAD-fmk、氯化锂（GSK3β 抑制剂）或组成型活性 β-连环蛋白，如 TCF 报告基因检测所示。此外，RKO 细胞用 TcdA 预孵育 12 小时也可减弱 Wnt3a 介导的 Wnt 信号激活，表明 Rho GTP 酶失活在该抑制中起重要作用。综上所述，这些发现表明 TcdA 对 Wnt 信号的抑制对于 TcdA 的抗增殖作用很重要。
