Cholesterol-dependent pore formation of Clostridium difficile toxin A
Cholesterol-dependent pore formation of Clostridium difficile toxin A
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DOI：
10.1074/jbc.m512720200
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发表时间：
2006-04-21
影响因子：
4.8
通讯作者：
Aktories, K
中科院分区：
文献类型：
--
作者：
Giesemann, T;Jank, T;Aktories, K
来自艰难梭菌的大梭菌细胞毒素毒素A和毒素B是已知引起腹泻相关性腹泻和伪膜性结肠炎的主要毒力因子。这两种毒素都是单葡糖基化的，从而破坏Rho家族的小GTP酶。最近，据报道，毒素B而不是毒素A在酸性条件下诱导靶细胞膜中的孔形成。在这里，我们重新评估了来自人结肠癌细胞中毒素A孔形成的数据。用天然或重组毒素A处理Rb-86（+）-负载细胞导致由酸性脉冲诱导的放射性阳离子外排增加。孔形成的功效依赖于膜胆固醇，因为用甲基-β-环糊精去除膜的胆固醇抑制Rb-86（+）流出，而胆固醇补充重建毒素A的孔形成活性。用毒素B获得了类似的结果。一致地，甲基-β-环糊精处理以浓度依赖性方式延迟细胞中毒。在黑色的脂质膜，毒素A诱导的离子渗透孔，仅在胆固醇的双层和在低pH值。相反，释放糖基磷脂酰肌醇锚定结构的磷脂酰肌醇特异性磷脂酶C治疗没有降低细胞对毒素A和B的敏感性。这些数据表明，在结肠细胞中，毒素A在酸性环境（例如内体）中诱导孔形成，类似于针对毒素B报道的，并且表明梭菌葡糖基化毒素的孔形成取决于胆固醇的存在。
The large clostridial cytotoxins toxin A and toxin B from Clostridium difficile are major virulence factors known to cause antibiotic-associated diarrhea and pseudomembranous colitis. Both toxins mono-glucosylate and thereby inactivate small GTPases of the Rho family. Recently, it was reported that toxin B, but not toxin A, induces pore formation in membranes of target cells under acidic conditions. Here, we reassessed data on pore formation of toxin A in cells derived from human colon carcinoma. Treatment of Rb-86(+)-loaded cells with native or recombinant toxin A resulted in an increased efflux of radioactive cations induced by an acidic pulse. The efficacy of pore formation was dependent on membrane cholesterol, since cholesterol depletion of membranes with methyl-beta-cyclodextrin inhibited Rb-86(+) efflux, and cholesterol repletion reconstituted pore-forming activity of toxin A. Similar results were obtained with toxin B. Consistently, methyl-beta-cyclodextrin treatment delayed intoxication of cells in a concentration-dependent manner. In black lipid membranes, toxin A induced ion-permeable pores only in cholesterol containing bilayers and at low pH. In contrast, release of glycosylphosphatidylinositol-anchored structures by phosphatidylinositol specific phospholipase C treatment did not reduce cell sensitivity toward toxins A and B. These data indicate that in colonic cells toxin A induces pore formation in an acidic environment (e.g. endosomes) similar to that reported for toxin B and suggest that pore formation by clostridial glucosylating toxins depends on the presence of cholesterol.