STRUCTURE AND MECHANISM OF CS1 PILUS ASSEMBLY

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。毛是毛发状的表面纤维，介导致病菌附着和定植到宿主组织。产肠毒素大肠杆菌（ETEC）的CS1菌毛系统是ETEC在人类肠道中建立和定植所必需的，导致腹泻病，这是第三世界国家婴儿和儿童发病率的重要原因。CS1在cooBACD操纵子上编码，由多个CooA亚基组成，这些亚基在远端末端具有粘附肠细胞所需的单个CooD亚基。此外，95 kda的外膜蛋白CooC和质周伴侣蛋白CooB是菌毛组装所必需的。具体的假设是，CS1菌毛的组装是由外膜蛋白CooC单独与CooB或CooA作为复合物结合后的构象变化引发的。该假设基于以下数据：1)CooC是一种完整的外膜蛋白；2)毛向细胞表面输出需要CooC的表达；3)CooB与CooC共同纯化并稳定CooC，防止其蛋白水解降解。因此，无论是CooB本身，还是作为CooA或CooD的复合物，都会引发CooC的构象变化，从而允许亚基转运到表面。我们研究的具体目的是：1。表征CooB的天然结构和寡聚状态。2. 定义CooC中的结构变化。3. 确定CooC在CooA聚合机理中的作用。从这些研究中获得的信息可能有助于设计针对ETEC感染提供保护的新疗法。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Pili are hair-like surface fibers that mediate attachment and colonization of pathogenic bacteria to host tissues. The CS1 pilus system from enterotoxigenic Escherichia coli (ETEC) is required for the establishment and colonization of ETEC to the human intestine, resulting in diarrheal disease that is a significant cause of morbidity in infants and children in third-world countries. CS1 is encoded on the cooBACD operon, and is composed of multiple CooA subunits that have at the distal tip the single subunit of CooD that is required for adherence to intestinal cells. Additionally, CooC, a 95-kDa outer membrane protein, and CooB, a periplasmic chaperone, are required for pilus assembly. The specific hypothesis is that the assembly of CS1 pili is initiated by a conformational change in the outer membrane protein CooC upon binding of CooB alone are as a complex with CooD or CooA. The hypothesis is based on data that indicate that 1) CooC is an integral outer membrane protein, 2) expression of CooC is required for export of pili to the cell surface and 3) CooB co-purifies with CooC and stabilizes CooC against proteolytic degradation. Therefore CooB, either by itself or as a complex with CooA or CooD, initiates a conformational change in CooC that allows transport of subunits to the surface. The specific aims of our research are: 1. Characterize the native structure and oligomerization state of CooB. 2. Define structural changes in CooC. 3. Determine the role of CooC in the mechanism of polymerization of CooA. Information obtained from these studies may be useful in the design of novel therapies that provide protection against ETEC infection.