Department of Microbial Pathogenesis, University of Maryland Dental School, Baltimore, MD 21201, USA
Pathog Dis. 2013 Feb;67(1):11-8. doi: 10.1111/2049-632X.12016. Epub 2013 Jan 14.
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.
TcdA 和 TcdB。毒素摄取和递呈的确切机制尚未明确，但目前的模型预测，半胱氨酸蛋白酶结构域（CPD）介导的葡糖基转移酶结构域（GTD）的自切割和释放对于中毒至关重要。为了确定 CPD 介导的切割对 TcdB 细胞毒性的重要性，我们生成了两种突变毒素-TcdB-C698S 和 TcdB-H653A-并检测了它们使细胞中毒的能力。CPD 突变体包含完整的 GTD，但缺乏半胱氨酸蛋白酶活性。这些突变体的效力降低，因为它们对细胞的作用延迟且需要比野生型 TcdB 更高的浓度。它们最终确实导致细胞圆化、Rho GTPases 的糖基化和凋亡，与 TcdB 引起的细胞圆化、糖基化和凋亡没有区别。尽管突变毒素引起完全的细胞圆化，但它们未能将 GTD 释放到细胞质中，而野生型 TcdB 则显示出 GTD 的显著自切割和释放。我们得出结论，半胱氨酸蛋白酶介导的 GTD 自切割和释放不是 TcdB 细胞毒性活性的先决条件，而是限制 Rho GTPase 糖基化的效力和速度。我们的发现修正并完善了 TcdB 作用模式和细胞内运输的现行模型。
