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首页> 外文期刊>Infection and immunity >Mutational Analysis of the Enzymatic Domain of Clostridium difficile Toxin B Reveals Novel Inhibitors of the Wild-Type Toxin
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Mutational Analysis of the Enzymatic Domain of Clostridium difficile Toxin B Reveals Novel Inhibitors of the Wild-Type Toxin

机译：艰难梭菌毒素B酶域的突变分析揭示了野生型毒素的新型抑制剂。

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Toxin B (TcdB), a major Clostridium difficile virulence factor, glucosylates and inactivates the small GTP-binding proteins Rho, Rac, and Cdc42. In the present study we provide evidence that enzymatically inactive fragments of the TcdB enzymatic domain are effective intracellular inhibitors of native TcdB. Site-directed and deletion mutants of the TcdB enzymatic region (residues 1 to 556), lacking receptor binding and cell entry domains, were analyzed for attenuation of glucosyltransferase and glucosylhydrolase activity. Five of six derivatives from TcdB1-556 were found to be devoid of enzymatic activity. In order to facilitate cell entry, mutants were genetically fused to lfn, which encodes the protective antigen binding region of anthrax toxin lethal factor and mediates the cell entry of heterologous proteins. In line with reduced enzymatic activity, the mutants also lacked cytotoxicity. Remarkably, pretreatment or cotreatment of cells with four of the mutants provided protection against the cytotoxic effects of native TcdB. Furthermore, a CHO cell line expressing enzymatically active TcdB1-556 was also protected by the mutant-derived inhibitors, suggesting that inhibition occurred at an intracellular location. Protection also was afforded by the inhibitor to cells treated with Clostridium sordellii lethal toxin (TcsL), which uses the same cosubstrate as TcdB but shares Rac only as a common substrate target. Finally, the inhibitor did not provide protection against Clostridium novyi alpha-toxin (Τcnα), which shares similar substrates with TcdB yet uses a different cosubstrate. This is the first report to demonstrate that the potential exists to inhibit toxins at their intracellular site of action by using inactive mutants.
机译：毒素B（TcdB）是主要的艰难梭菌毒力因子，它使葡萄糖基化并使小GTP结合蛋白Rho，Rac和Cdc42失活。在本研究中，我们提供证据表明，TcdB酶促结构域的酶促失活片段是天然TcdB的有效细胞内抑制剂。分析缺乏受体结合和细胞进入结构域的TcdB酶促区域（残基1至556）的定点和缺失突变体，以降低葡萄糖基转移酶和葡萄糖基水解酶的活性。发现来自TcdB 1-556 的六个衍生物中有五个没有酶促活性。为了促进细胞进入，将突变体与 lfn 进行基因融合，该基因编码炭疽毒素致死因子的保护性抗原结合区并介导异源蛋白质的细胞进入。与降低的酶活性一致，突变体也缺乏细胞毒性。值得注意的是，用四种突变体对细胞进行预处理或共处理可提供针对天然TcdB的细胞毒性作用的保护作用。此外，表达酶活性的TcdB 1-556 的CHO细胞系也受到突变来源的抑制剂的保护，这表明抑制作用发生在细胞内。抑制剂还对用 Clostridium sordellii 致死毒素（TcsL）处理的细胞提供了保护，该细胞使用与TcdB相同的共底物，但仅将Rac用作共同的底物靶标。最终，该抑制剂没有提供针对<新>梭状芽孢杆菌α-毒素（Τcnα）的保护，后者与TcdB具有相似的底物，但使用不同的共底物。这是第一个证明通过使用非活性突变体在其细胞内作用位点抑制毒素存在的潜力的报告。

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