Membrane Translocation of Binary Actin-ADP-Ribosylating Toxins from Clostridium difficile and Clostridium perfringens Is Facilitated by Cyclophilin A and Hsp90
Membrane Translocation of Binary Actin-ADP-Ribosylating Toxins from Clostridium difficile and Clostridium perfringens Is Facilitated by Cyclophilin A and Hsp90
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DOI：
10.1128/iai.05372-11
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发表时间：
2011-10-01
影响因子：
3.1
通讯作者：
Barth, Holger
中科院分区：
文献类型：
--
作者：
Kaiser, Eva;Kroll, Claudia;Barth, Holger
艰难梭菌的一些超强毒株产生二元肌动蛋白-ADP-核糖基化毒素C。艰难梭菌转移酶（CDT）以及Rho-葡糖基化毒素A和B。有人认为，CDT的存在增加了C。艰难梭菌相关疾病，包括伪膜性结肠炎。CDT含有结合和易位组分CDTb，其介导单独的酶组分CDTa转运到靶细胞的胞质溶胶中，其中CDTa修饰肌动蛋白。在这里，我们研究了细胞CDT摄取的机制，发现巴弗洛霉素A1保护培养的上皮细胞免于CDT中毒，这意味着CDTa从酸化的内体囊泡易位到胞质溶胶中。同样，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胞质溶胶中。Radicicol和环孢素A，热休克蛋白Hsp 90和亲环素的抑制剂，分别保护细胞免于CDT中毒，但不保护细胞免于毒素A和B中毒。此外，这两种抑制剂阻断了CDTa的pH依赖性膜转位，强烈表明Hsp 90和亲环蛋白对该过程至关重要。相反，抑制剂不干扰ADP-核糖基转移酶活性，受体结合，或内吞作用的毒素。我们获得了与产气荚膜梭菌密切相关的iota毒素相当的结果。此外，CDTa和Ia，iota毒素的酶组分，在体外特异性结合固定化Hsp 90和亲环素A。结合我们最近获得的关于C.肉毒杆菌，这些结果意味着一个共同的热休克蛋白90/亲环素A依赖性易位机制的家庭二元肌动蛋白-ADP-核糖基化毒素。
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.