Structure & Function of Clostridium difficile Type IV Pili

 描述(申请人提供)：艰难梭菌是一种形成芽胞的厌氧革兰氏阳性杆菌，可导致人类从轻度腹泻到伪膜性结肠炎等各种胃肠道疾病，严重时可导致中毒性巨结肠，这是一种急性、有时是致命的结肠扩张形式。艰难梭菌结肠炎的发病率、严重程度和死亡率在过去20年中显著增加。然而，关于艰难梭菌与人类宿主之间相互作用的知识几乎是不存在的，严重阻碍了艰难梭菌疾病预防、控制和治疗的新方法的发展。IV型菌毛是由许多细菌产生的菌毛表面附属物，在细胞黏附、定植、抽动运动、生物膜形成和水平基因转移中发挥关键作用。它们往往对毒力至关重要，有些已经成功地作为疫苗开发出来。IV型菌毛在革兰氏阴性菌中广泛存在，但对革兰氏阳性菌的这些菌毛几乎一无所知。然而，IV型菌毛蛋白基因存在于梭状芽孢杆菌属所有成员的基因组中，所有艰难梭菌株都编码一整套T4P生物发生机械组件和数量可变的IV型菌毛蛋白。我们推测，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，直接介导人类细胞的黏附，这对定植和毒力都很重要。因此，我们试图将艰难梭菌IV型菌毛结构定义为单独的蛋白质成分和组装的超分子附属物，并通过追求以下特定目标来鉴定它们的宿主细胞受体：(1)确定艰难梭菌IV型菌毛蛋白的原子结构；(2)确定艰难梭菌IV型菌毛的组成和结构；(3)鉴定艰难梭菌IV型菌毛特异参与的宿主分子。我们提出的研究艰难梭菌IV型菌毛结构和功能的综合方法将利用多种分辨率的蛋白质结构测定方法，包括X射线结晶学、核磁共振光谱、小角X射线散射和冷冻电子显微镜，以及基于最先进的质谱学方法来定义多组分蛋白质组件的组成，绘制蛋白质-蛋白质界面图，并识别新的受体分子。为了确保我们提议的研究的成功，我们组建了一支优秀的研究团队，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具有广泛的专业知识。

 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.