The glucosyltransferase activity of C. difficile Toxin B is required for disease pathogenesis.
The glucosyltransferase activity of C. difficile Toxin B is required for disease pathogenesis.
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DOI：
10.1371/journal.ppat.1008852
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发表时间：
2020-09
期刊：
影响因子：
6.7
通讯作者：
Melnyk RA
中科院分区：
文献类型：
--
作者：
Bilverstone TW;Garland M;Cave RJ;Kelly ML;Tholen M;Bouley DM;Kaye P;Minton NP;Bogyo M;Kuehne SA;Melnyk RA
艰难梭菌毒素 B (TcdB) 的葡萄糖基转移酶结构域对 Rho 家族 GTP 酶的酶促失活会在细胞中产生各种致病作用，这些作用通常被认为是与艰难梭菌感染 (CDI) 相关的疾病症状的原因。最近的体外研究表明，在某些情况下，TcdB 可以诱导与葡萄糖基转移酶 (GT) 活性无关的细胞毒性，这使人们对 GT 活性的确切作用产生疑问。在这里，为了确定 GT 活性在 CDI 疾病发病机制中的重要性，我们生成了第一个描述的产生葡萄糖基转移酶缺陷（GT 缺陷）毒素的艰难梭菌突变株。使用等位基因交换（AE）技术，我们首先删除了艰难梭菌 630Δerm 中的 tcdA，随后在 TcdB 的 GT 结构域中引入了失活的 D270N 替换。为了研究 GT 活性在体内的作用，我们在两种不同的 CDI 发病机制动物模型中测试了每种菌株。在非致死性小鼠感染模型中，与野生型和 630ΔermΔtcdA (ΔtcdA) 菌株中观察到的严重盲肠炎症相比，GT 缺陷突变体在宿主组织中引起的病理最小化。在更敏感的 CDI 仓鼠模型中，野生型或 ΔtcdA 组的仓鼠在 4 天内死于暴发性感染，而所有感染 GT 缺陷突变体的仓鼠都在 10 天的感染期中存活下来，没有 CDI 的主要症状或盲肠炎症的证据。这些数据表明 GT 活性对于疾病发病机制是不可或缺的，并重申了其在疾病中的核心作用及其作为小分子抑制治疗靶点的重要性。治疗艰难梭菌感染（CDI）需要新型非抗生素疗法。一类新兴的有前景的 CDI 疗法是抗毒剂，它可以阻断艰难梭菌毒素 B (TcdB) 的作用，而 TcdB 是毒力的主要决定因素。为了开发此类治疗方法，必须确定和验证分子靶点和机制。历史上，葡萄糖基转移酶结构域（GTD）由于其对疾病发病机制的重要性而成为理想的靶标。然而，利用重组 TcdB 生产的最新进展的研究揭示了体外高浓度应用时不依赖于 GTD 的毒性机制，从而对 GTD 的作用提出了质疑。在这里，我们生成了首次报道的表达葡萄糖基转移酶缺陷型 TcdB 的艰难梭菌突变株。其应用表明GTD对于小鼠和仓鼠的疾病是必需的，从而使GTD重新成为小分子抑制剂（SMI）开发的理想候选者。
Enzymatic inactivation of Rho-family GTPases by the glucosyltransferase domain of Clostridioides difficile Toxin B (TcdB) gives rise to various pathogenic effects in cells that are classically thought to be responsible for the disease symptoms associated with C. difficile infection (CDI). Recent in vitro studies have shown that TcdB can, under certain circumstances, induce cellular toxicities that are independent of glucosyltransferase (GT) activity, calling into question the precise role of GT activity. Here, to establish the importance of GT activity in CDI disease pathogenesis, we generated the first described mutant strain of C. difficile producing glucosyltransferase-defective (GT-defective) toxin. Using allelic exchange (AE) technology, we first deleted tcdA in C. difficile 630Δerm and subsequently introduced a deactivating D270N substitution in the GT domain of TcdB. To examine the role of GT activity in vivo, we tested each strain in two different animal models of CDI pathogenesis. In the non-lethal murine model of infection, the GT-defective mutant induced minimal pathology in host tissues as compared to the profound caecal inflammation seen in the wild-type and 630ΔermΔtcdA (ΔtcdA) strains. In the more sensitive hamster model of CDI, whereas hamsters in the wild-type or ΔtcdA groups succumbed to fulminant infection within 4 days, all hamsters infected with the GT-defective mutant survived the 10-day infection period without primary symptoms of CDI or evidence of caecal inflammation. These data demonstrate that GT activity is indispensable for disease pathogenesis and reaffirm its central role in disease and its importance as a therapeutic target for small-molecule inhibition. Novel non-antibiotic therapies are required for the treatment of Clostridioides difficile infection (CDI). An emerging class of promising therapeutics for CDI are antivirulence agents that block the actions of C. difficile Toxin B (TcdB), the primary determinant of virulence. In order to develop such treatments, molecular targets and mechanisms must be identified and validated. Historically the glucosyltransferase domain (GTD) represented an ideal target owing to its perceived importance for disease pathogenesis. However, studies capitalizing on recent advances in recombinant TcdB production have unveiled GTD-independent mechanisms of toxicity when applied at high concentrations in vitro, thus questioning the role of the GTD. Here we generate the first-reported mutant strain of C. difficile expressing glucosyltransferase-defective TcdB. Application thereof demonstrates that the GTD is essential for disease in mice and hamsters, thus reoffering the GTD as an ideal candidate for small-molecule inhibitor (SMI) development.
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影响因子：
6.7
作者：
Chumbler NM;Farrow MA;Lapierre LA;Franklin JL;Haslam DB;Goldenring JR;Lacy DB
通讯作者：
Lacy DB
影响因子：
3.7
作者：
Ng YK;Ehsaan M;Philip S;Collery MM;Janoir C;Collignon A;Cartman ST;Minton NP
通讯作者：
Minton NP
影响因子：
3.5
作者：
Pathak, Ashish K.;Pathak, Vibha;Reynolds, Robert C.
通讯作者：
Reynolds, Robert C.
影响因子：
--
作者：
Bouillaut, Laurent;McBride, Shonna M;Sorg, Joseph A
通讯作者：
Sorg, Joseph A
DOI：
10.1056/nejmra0707500
发表时间：
2008-10-30
期刊：
The New England journal of medicine
影响因子：
--
作者：
Kelly, Ciaran P;LaMont, J Thomas
通讯作者：
LaMont, J Thomas