Cytotoxicity of Clostridium difficile toxin B does not require cysteine protease-mediated autocleavage and release of the glucosyltransferase domain into the host cell cytosol
Cytotoxicity of Clostridium difficile toxin B does not require cysteine protease-mediated autocleavage and release of the glucosyltransferase domain into the host cell cytosol
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DOI：
10.1111/2049-632x.12016
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发表时间：
2013-02-01
影响因子：
3.3
通讯作者：
Feng, Hanping
中科院分区：
文献类型：
--
作者：
Li, Shan;Shi, Lianfa;Feng, Hanping
艰难梭菌的毒力需要分泌两种外毒素：TcdA和TcdB。毒素摄取和递送的精确机制尚不明确，但目前的模型预测半胱氨酸蛋白酶结构域（CPD）介导的葡萄糖基转移酶结构域（GTD）的自切割和释放对中毒至关重要。为了确定CPD介导的切割对TcdB细胞毒性的重要性，我们产生了两种突变毒素-TcdB-C698 S和TcdB-H653 A-并测定了它们使细胞中毒的能力。CPD突变体包括完整的GTD，但缺乏半胱氨酸蛋白酶活性。突变体具有降低的效力，因为它们对细胞的作用被延迟并且需要比野生型TcdB更高的浓度。它们最终导致细胞变圆、Rho GTP酶的糖基化和与TcdB引起的细胞凋亡难以区分的细胞凋亡。虽然突变体毒素造成了一个完整的细胞变圆，他们未能释放到胞质溶胶中的GTD，而野生型TcdB显示显着的自切割和释放的GTD。我们的结论是，半胱氨酸蛋白酶介导的自切割和释放的GTD是不是TcdB的细胞毒活性的先决条件，而是限制了Rho GTdR糖基化的效力和速度。我们的研究结果修改和完善了TcdB的作用模式和细胞运输的当前模型。
Clostridium difficile virulence requires secretion of two exotoxins: TcdA and TcdB. The precise mechanism of toxin uptake and delivery is undefined, but current models predict that the cysteine protease domain (CPD)-mediated autocleavage and release of glucosyltransferase domain (GTD) are crucial for intoxication. To determine the importance of CPD-mediated cleavage to TcdB cytotoxicity, we generated two mutant toxins - TcdB-C698S and TcdB-H653A - and assayed their abilities to intoxicate cells. The CPD mutants include an intact GTD but lack the cysteine protease activity. The mutants had reduced potency in that their effect on cells was delayed and required higher concentrations than wild-type TcdB. They did eventually cause cell rounding, glucosylation of Rho GTPases, and apoptosis that was indistinguishable from that caused by TcdB. Although the mutant toxins caused a complete cell rounding, they failed to release their GTD into cytosol, whereas wild-type TcdB displayed significant autocleavage and release of GTD. We conclude that the cysteine protease-mediated autocleavage and release of GTD is not a prerequisite for the cytotoxic activity of TcdB, but rather limits the potency and speed of Rho GTPase glucosylation. Our findings revise and refine the current model for the mode of the action and cellular trafficking of TcdB.