Institute of Pharmacology and Toxicology, University of Ulm Medical Center, Ulm, Germany.
Infect Immun. 2011 Oct;79(10):3913-21. doi: 10.1128/IAI.05372-11. Epub 2011 Jul 18.
Some hypervirulent strains of Clostridium difficile produce the binary actin-ADP-ribosylating toxin C. difficile transferase (CDT) in addition to Rho-glucosylating toxins A and B. It has been suggested that the presence of CDT increases the severity of C. difficile-associated diseases, including pseudomembranous colitis. CDT contains a binding and translocation component, CDTb, that mediates the transport of the separate enzyme component CDTa into the cytosol of target cells, where CDTa modifies actin. Here we investigated the mechanism of cellular CDT uptake and found that bafilomycin A1 protects cultured epithelial cells from intoxication with CDT, implying that CDTa is translocated from acidified endosomal vesicles into the cytosol. Consistently, CDTa is translocated across the cytoplasmic membranes into the cytosol when cell-bound CDT is exposed to acidic medium. Radicicol and cyclosporine A, inhibitors of the heat shock protein Hsp90 and cyclophilins, respectively, protected cells from intoxication with CDT but not from intoxication with toxins A and B. Moreover, both inhibitors blocked the pH-dependent membrane translocation of CDTa, strongly suggesting that Hsp90 and cyclophilin are crucial for this process. In contrast, the inhibitors did not interfere with the ADP-ribosyltransferase activity, receptor binding, or endocytosis of the toxin. We obtained comparable results with the closely related iota-toxin from Clostridium perfringens. Moreover, CDTa and Ia, the enzyme component of iota-toxin, specifically bound to immobilized Hsp90 and cyclophilin A in vitro. In combination with our recently obtained data on the C2 toxin from C. botulinum, these results imply a common Hsp90/cyclophilin A-dependent translocation mechanism for the family of binary actin-ADP-ribosylating toxins.
一些产毒力较强的艰难梭菌除了产生 Rho 葡糖基化毒素 A 和 B 之外，还会产生二聚体肌动蛋白-ADP- 核糖基转移酶 C. 艰难梭菌转移酶（CDT）。据推测，CDT 的存在会增加艰难梭菌相关疾病的严重程度，包括伪膜性结肠炎。CDT 包含一个结合和易位组件 Cdtb，它介导独立的酶组件 Cdta 进入靶细胞的细胞质，在那里 Cdta 修饰肌动蛋白。在这里，我们研究了细胞 CDT 摄取的机制，并发现巴弗洛霉素 A1 可保护培养的上皮细胞免受 CDT 的中毒，这意味着 Cdta 是从酸化的内体小泡易位到细胞质中的。一致地，当细胞结合的 CDT 暴露于酸性介质时，Cdta 穿过细胞质膜易位到细胞质中。热休克蛋白 Hsp90 和亲环蛋白的抑制剂，即雷迪霉素和环孢菌素 A，可保护细胞免受 CDT 中毒，但不能免受毒素 A 和 B 的中毒。此外，两种抑制剂均阻断了 CDTa 的 pH 依赖性膜易位，强烈表明 Hsp90 和亲环蛋白对于该过程至关重要。相反，抑制剂不干扰毒素的 ADP- 核糖基转移酶活性、受体结合或内吞作用。我们用来自产气荚膜梭菌的密切相关的iota 毒素获得了类似的结果。此外，Cdta 和 Ia，即 iota 毒素的酶组件，特异性地与固定化 Hsp90 和亲环蛋白 A 结合。结合我们最近关于肉毒梭菌 C2 毒素的数据，这些结果表明家族的二聚体肌动蛋白-ADP- 核糖基转移酶具有共同的 Hsp90/亲环蛋白 A 依赖性易位机制。
