Structure & Function of Clostridium difficile Type IV Pili

结构

• 批准号: 10087197
• 负责人: ERIC JOHN SUNDBERG
• 金额: $ 37.9万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-03-05 至 2021-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10087197
• 关键词: Structure; Function; Clostridium; difficile; Type

 DESCRIPTION (provided by applicant): Clostridium difficile is a spore-forming anaerobic Gram-positive bacillus that causes gastrointestinal illnesses in humans ranging from mild diarrhea to pseudomembranous colitis, which in severe cases can lead to toxic megacolon, an acute and sometimes lethal form of colonic distension. The incidence, severity and mortality of C. difficile colitis have increased significantly over the last two decades. However, knowledge concerning interactions between C. difficile bacteria and the human host is virtually nonexistent, severely impeding the development of new approaches to the prevention, control, and treatment of C. difficile disease. Type IV pili are fimbrial surface appendages produced by many bacteria that play critical roles in cellular adhesion, colonization, twitching motility, biofilm formation,and horizontal gene transfer. They are often essential for virulence and some have been successfully developed as vaccines. Type IV pili have been characterized extensively in Gram-negative bacteria, but nearly nothing is known about these pili from Gram-positive bacteria. Type IV pilin genes, though, are present in the genomes of all members of the genus Clostridium and all C. difficile strains encode complete sets of T4P biogenesis machinery components and a variable number of Type IV pilin proteins. We hypothesize that C. difficile Type IV pili, through structural features distinct from those of Gram-negative Type IV pili, directy mediate human cell adherence that is important for both colonization and virulence. Accordingly, we seek to define C. difficile Type IV pilin structures, both as individual protein components and assembled supramolecular appendages, and to identify their human host cell receptors, by pursuing the following Specific Aims: (1) to determine the atomic structures of individual C. difficile Type IV pilin proteins; (2) to define the composition and architecture of C. difficile Tye IV pili; and (3) to identify host molecules engaged specifically by C. difficile Type IV pili. The comprehensive approach that we propose to investigate the structure and function of C. difficile Type IV pili will utilize protein structure determination methods throughout a broad range of resolutions, including X-ray crystallography, NMR spectroscopy, small-angle X-ray scattering and cryo-electron microscopy, as well as state-of-the-art mass spectrometry-based approaches to define the composition of multi-component protein assemblies, map protein-protein interfaces and identify novel receptor molecules. To assure the success of our proposed studies, we have assembled a team of outstanding investigators with extensive expertise in these experimental techniques, C. difficile biology and Type IV pilus structure and function.

 描述（由申请人提供）：艰难梭菌是一种形成孢子的厌氧革兰氏阳性杆菌，可引起人类胃肠道疾病，从轻度腹泻到伪膜性结肠炎，在严重的情况下可导致中毒性巨结肠，这是一种急性且有时致命的结肠炎。过去二十年来，艰难梭菌结肠炎的发病率、严重程度和死亡率显着增加。艰难梭菌和人类宿主之间几乎不存在，这严重阻碍了预防、控制和治疗艰难梭菌疾病的新方法的开发。 IV 型菌毛是由许多细菌产生的菌毛表面附属物，在细胞粘附中发挥着关键作用。它们的定植、抽搐运动、生物膜形成和水平基因转移通常对于毒力至关重要，并且某些已成功开发为疫苗，已在革兰氏阴性细菌中进行了专门表征，但几乎没有。然而，我们对这些来自革兰氏阳性细菌的菌毛一无所知，但它们存在于梭菌属所有成员的基因组中，并且所有艰难梭菌菌株都编码完整的 T4P 生物发生机制组件和数量可变的组件。我们发现，艰难梭菌 IV 型菌毛通过与革兰氏阴性 IV 型菌毛不同的结构特征，直接介导人类细胞粘附，这对于定植和毒力都很重要。因此，我们试图通过追求以下具体目标来定义艰难梭菌 IV 型菌毛蛋白结构，包括单独的蛋白质成分和组装的超分子附属物，并鉴定其人类宿主细胞受体：（1）确定个体的原子结构艰难梭菌 IV 型菌毛蛋白；(2) 定义艰难梭菌 IV 型菌毛的组成和结构；以及 (3) 鉴定艰难梭菌 IV 型菌毛特异性参与的宿主分子。我们建议研究艰难梭菌 IV 型菌毛的结构和功能，将利用各种分辨率的蛋白质结构测定方法，包括 X 射线晶体学、核磁共振波谱、小角 X 射线散射和冷冻电子显微镜以及最先进的基于质谱的方法来定义多组分蛋白质组装体的组成，绘制蛋白质-蛋白质界面并识别新的受体分子，以确保我们提出的成功。在这些研究中，我们组建了一支由优秀研究人员组成的团队，他们在这些实验技术、艰难梭菌生物学和 IV 型菌毛结构和功能方面拥有丰富的专业知识。