Structural mechanisms of Clostridium difficile pathogenesis

艰难梭菌发病机制的结构机制

• 批准号: 9212765
• 负责人: Dana Borden Lacy
• 金额: $ 39.43万
• 依托单位: VANDERBILT UNIVERSITY MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-05-15 至 2021-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9212765
• 关键词: Anaerobic Bacteria; Antibiotics; Antibodies; Apoptosis; Bacteria; Binding; Binding Sites; CRISPR/Cas technology; Cell physiology; Cells; Cellular Assay; Clostridium difficile; Colon; Complex; Crystallization; Cytosol; Data; Development; Diarrhea; Disease; Electron Microscopy; Endosomes; Epithelial Cells; Event; Family; Fluorescence; Funding; Genetic Variation; Glucosyltransferase; Goals; Guanosine Triphosphate Phosphohydrolases; Homologous Protein; Hospitals; Human; Human poliovirus; Hybrids; Hydrophobicity; Incidence; Individual; Infection; Inflammation; Integration Host Factors; Intoxication; Label; Lead; Measurement; Mediating; Membrane; Modeling; Molecular; Mus; Mutagenesis; Mutation; NADPH Oxidase; Necrosis; Normal tissue morphology; Pathogenesis; Pathology; Physiological; Prevention; Production; Proteins; Pseudomembranous Colitis; Public Health; Publishing; Reactive Oxygen Species; Reproduction spores; Research; Role; Series; Severities; Structure; Testing; Therapeutic; Therapeutic Intervention; Tissues; Toxic Megacolon; Toxin; United States; Vaccine Antigen; Vaccines; Variant; Viral Proteins; Virulence Factors; X-Ray Crystallography; alpha Toxin; alpha helix; cost; crosslink; cytotoxicity; design; electron tomography; experimental study; follow-up; human disease; improved; mouse model; new therapeutic target; novel therapeutics; poliovirus receptor; programs; public health relevance; receptor; receptor mediated endocytosis; rho; scaffold; therapeutic target; tool

 DESCRIPTION (provided by applicant): Clostridium difficile is a gram-positive, spore-forming anaerobe that infects the colon, causing a range of human disease including diarrhea, pseudomembranous colitis, and toxic megacolon. The incidence, severity, and costs associated with C. difficile infection (CDI) are increasing, making C. difficile a significant public health concern. The principle virulence factors in C. difficile pathogenesis are TcdA and TcdB, two large homologous toxins capable of entering and modifying multiple targets within eukaryotic host cells. This proposal is designed to follow up two important discoveries made in the previous funding cycle, a crystal structure of TcdA and the cellular receptor for TcdB. In Aim 1, we will use the structure of the TcdA delivery domain as a framework for understanding toxin pore formation within the host endosomal membrane. A hybrid approach of electron microscopy, crosslinking, and fluorescence intensity measurements will generate a structural view of the toxin pore. In Aim 2, we will define the interaction of TcdB with its receptor, PVRL3, using a hybrid of electron microscopy, mutagenesis, quantitative binding studies, and X-ray crystallography. In Aim 3, we will evaluate the relevance of the TcdB-PVRL3 interaction in mouse models of intoxication and infection. Results from these aims will provide a molecular understanding of the events that allow toxins access to the host cell and a framework for therapeutic intervention.

 描述（由申请人提供）：艰难梭菌是一种革兰氏阳性、产芽孢厌氧菌，可感染结肠，引起一系列人类疾病，包括腹泻、伪膜性结肠炎和中毒性巨结肠。与艰难梭菌感染 (CDI) 相关的发病率、严重程度和成本不断增加，使得艰难梭菌成为重大的公共卫生问题。艰难梭菌发病机制中的主要毒力因子是 TcdA 和 TcdB，这两种大的同源毒素能够进入并修饰真核宿主细胞内的多个靶标。该提案旨在跟进上一个资助周期中的两个重要发现，即 TcdA 的晶体结构和 TcdB 的细胞受体。在目标 1 中，我们将使用 TcdA 传递结构域的结构作为理解宿主内体膜内毒素孔形成的框架。电子显微镜、交联和荧光强度测量的混合方法将生成毒素孔的结构视图。在目标 2 中，我们将综合使用电子显微镜、诱变、定量结合研究和 X 射线晶体学来定义 TcdB 与其受体 PVRL3 的相互作用。在目标 3 中，我们将评估 TcdB-PVRL3 相互作用在小鼠中毒和感染模型中的相关性。这些目标的结果将提供对允许毒素进入宿主细胞的事件的分子理解以及治疗干预的框架。