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

基于结构的高毒力细菌菌株中艰难梭菌毒素 (CDT) 的靶向

• 批准号: 10455150
• 负责人: David Joseph Weber
• 金额: $ 52.18万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-08-06 至 2023-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10455150
• 关键词: 3-Dimensional; Address; Adenosine Diphosphate Ribose; Amino Acids; Anisotropy; Antibiotics; Area; Bacteria; Bacterial Infections; Binding; Biological Assay; CD44 gene; Cells; Chemicals; Classification; Clostridium difficile; Colonoscopy; Complex; Computer software; Corynebacterium diphtheriae; Cryoelectron Microscopy; Cytoplasm; Dangerousness; Data; Data Analyses; Detection; Development; Diarrhea; Diphtheria Toxin; Enterotoxins; Enzymes; Future; G Actin; Generations; Goals; Gold; Healthcare; Infection; Journals; Laboratories; Length; Measures; Medical; Membrane; Methods; Modeling; Modification; Molecular; Mutation; Peer Review; Pharmaceutical Preparations; Physiological; Preparation; Process; Proteins; Pseudomonas aeruginosa; Publications; Receptor Cell; Recurrence; Reporting; Research; Resolution; Roentgen Rays; Site; Site-Directed Mutagenesis; Structure; System; Targeted Toxins; Therapeutic; Toxic effect; Toxin; Validation; Work; X-Ray Crystallography; base; biophysical properties; cytolethal distending toxin; cytotoxic; density; drug development; drug discovery; experimental study; fecal transplantation; instrumentation; interest; mutant; programs; protein structure; public health priorities; receptor; receptor binding; response; structural biology; therapeutic target; treatment strategy

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.

摘要 – 针对艰难梭菌感染 (CDI) 具有挑战性，因为治疗方法仅限于 少量抗生素、结肠镜检查和实验方法（即粪便移植），并且有 复发率高得令人难以接受，尤其是 NAP1/P1/027 等高毒力菌株。 高毒力菌株的问题在于它们具有一种称为艰难梭菌毒素 (CDT) 的二元毒素，此外 肠毒素 TsdA 和 TsdB CDT 二元毒素具有称为 CDTa 的酶成分和 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的结果。 通过 CDTa 将 NAD 的 ADP-核糖部分转移至其生理底物宿主 G-肌动蛋白。 CDTa 通过二元毒素 CDTb 的成孔成分进入细胞质。 TcdA/TcdB 存在，没有针对 CDT 的治疗方法以及 CDTa/CDTb 二元的作用机制 在我们的实验室中，与 CDI 高毒力菌株相关的毒素尚不清楚。 正在通过冷冻电镜方法使用活性和完全表征 CDTa/CDTb 二元复合物 处理 CDTa 和 CDTb 的全长结构（目标 1）。 使用 NMR 和 X 射线进行涉及二元毒素特定域的结构和动态研究 晶体学（目标 1）中，计划对与宿主细胞参与相关的复合物进行研究。 包括膜样介质中的 CDTb 和 CDTa/CDTb 复合物，并与衍生的构建体结合 来自宿主细胞受体（即 CD44、LSR）的结构将一起用于定义 CDT 上的区域。 作为一种治疗方法，对于参与宿主细胞并提供阻断这一过程所需的信息非常重要 因为其他传染性细菌利用类似的“III 型”系统来传递 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）， 预计这里完成的 CDI 研究可能有利于高毒力梭菌的治疗策略。 艰难梭菌以及其他危险的细菌感染。

项目成果

1HN, 13C, and 15N resonance assignments of the Clostridioides difficile receptor binding domain 2 (CDTb, residues 757-876).

艰难梭菌受体结合域 2（CDTb，残基 757-876）的 1HN、13C 和 15N 共振归属。

• 发表时间: 2021-04
• 影响因子: 0.9
• 作者: Cook, Mary E;Varney, Kristen M;Godoy;Weber, David J
• 通讯作者: Weber, David J

Computer-Aided Drug Design: An Update.

计算机辅助药物设计：更新。

• DOI: 10.1007/978-1-0716-2855-3_7
• 发表时间: 2024-09-14
• 期刊: Methods in molecular biology
• 作者: Wenbo Yu;David J. Weber;Ale;er D. MacKerell;er
• 通讯作者: er

The Importance of Therapeutically Targeting the Binary Toxin from Clostridioides difficile.

治疗靶向艰难梭菌二元毒素的重要性。

• 发表时间: 2021-03-13
• 影响因子: 5.6
• 作者: Abeyawardhane, Dinendra L;Godoy;Adipietro, Kaylin A;Varney, Kristen M;Rustandi, Richard R;Pozharski, Edwin;Weber, David J
• 通讯作者: Weber, David J

Binding and Functional Folding (BFF): A Physiological Framework for Studying Biomolecular Interactions and Allostery.

结合和功能折叠（BFF）：研究生物分子相互作用和变构的生理框架。

• 发表时间: 2022-12-15
• 影响因子: 5.6
• 作者: Young, Brianna D;Cook, Mary E;Costabile, Brianna K;Samanta, Riya;Zhuang, Xinhao;Sevdalis, Spiridon E;Varney, Kristen M;Mancia, Filippo;Matysiak, Silvina;Lattman, Eaton;Weber, David J
• 通讯作者: Weber, David J