Structure-based targeting of the C. difficile toxin (CDT) from hypervirulent bacterial strains

摘要-靶向艰难梭菌感染（CDI）是具有挑战性的，因为治疗仅限于 少量抗生素、结肠镜检查和实验方法（即粪便移植）， 不可接受的高复发率，特别是对于高毒力菌株如NAP 1/P1/027。一个严重 高毒力菌株的问题是它们具有称为C.艰难梭菌毒素（CDT），此外 肠毒素TsdA和TsdB。CDT二元毒素具有称为CDTa的酶组分和称为CDTa的酶组分。 称为CDTb的孔形成或递送亚基。对宿主细胞的细胞毒性反应是催化作用的结果 通过CDTa将NAD的ADP-核糖部分转移到宿主G-肌动蛋白（其生理底物）。交付 CDTa进入细胞质是通过二元毒素的成孔组分CDTb。虽然治疗方案 存在TcdA/TcdB，没有靶向CDT的治疗和CDTa/CDTb二元的作用机制 与CDI的高毒力菌株相关的毒素还不清楚。在我们的实验室里， 一种方法正在通过cryoEM来表征CDTa/CDTb二元复合物， 处理CDTa和CDTb的全长构建体（Aim 1）。这将是随后更详细 利用核磁共振和X射线对二元毒素的特定结构域进行结构和动力学研究 晶体学（目标1）。在目标2中，计划研究与宿主细胞接合相关的复合物， 包括CDTb和CDTa/CDTb复合物在膜样介质中的那些，并结合到衍生的构建体， 来自宿主细胞受体（即CD 44、LSR）。这些结构将一起用于在CDT上定义区域 对于接合宿主细胞并提供阻断该过程所必需的信息是重要的， approach.因为其他感染性细菌利用类似的“III型”系统来递送ADP- 核糖基化毒素（即白喉棒状杆菌、白喉毒素、铜绿假单胞菌等）， 预期在此完成的CDI研究可有益于高毒力C的治疗策略。 艰难梭菌以及其他危险的细菌感染。

Abstract – Targeting Clostridium difficile infection (CDI) is challenging because treatments are limited to a small number of antibiotics, colonoscopy, and experimental methods (i.e. fecal transplant), and there are unacceptably high recurrence rates, particularly with hypervirulent strains such as NAP1/P1/027. One serious problem with hypervirulent strains is that they have a binary toxin termed the C. difficile toxin (CDT), in addition to the enterotoxins TsdA and TsdB. The CDT binary toxin has an enzymatic component, termed CDTa, and a pore-forming or delivery subunit termed CDTb. The cytotoxic response to host cells is the result of a catalytic transfer of the ADP-ribose moiety of NAD to host G-actin, its physiological substrate, via CDTa. Delivery of CDTa to the cytoplasm is via the pore-forming component of the binary toxin, CDTb. While treatment options exist for TcdA/TcdB, there is no therapy to target CDT and the mechanism of action for the CDTa/CDTb binary toxin associated with the hypervirulent strains of CDI is not well understood. In our labs, a structural biology approach is underway via cryoEM to characterize the CDTa/CDTb binary complex using active and fully processed full-length constructs of both CDTa and CDTb (Aim 1). This will be followed by more detailed structural and dynamic studies involving specific domains of the binary toxins using NMR and X-ray crystallography (Aim 1). In Aim 2, studies of complexes relevant to host cell engagement are planned to include those of CDTb and the CDTa/CDTb complex in membrane-like media and bound to constructs derived from host cell receptors (i.e. CD44, LSR). Together the structures will be used to define regions on CDT important for engaging the host cell and provide information necessary to block this process, as a therapeutic approach. Because other infectious bacteria make use of similar “Type III” systems for delivering ADP- ribosylating toxins (i.e. Corynebacterium diphtheriae, diphtheria toxin, Pseudomonas aeruginosa, and others), it is anticipated that the research completed here for CDI could benefit treatment strategies for hypervirulent C. difficile as well as for other dangerous bacterial infections.