Structural Microbiology of Type IV Pilus Retraction

描述（由申请人提供）：IV型毛是细胞外的细菌细丝，在许多植物、动物和人类感染（包括由脑膜炎奈瑟菌引起的细菌性脑膜炎，由淋病奈瑟菌引起的淋病，以及由铜绿假单胞菌引起的囊性纤维化患者的肺部感染）中附着在表面和发病机制所必需的。通过细胞内atp酶PilT的作用，毛被缩回到它们出现的细胞中。收缩导致表面运动并激活真核靶细胞的信号通路。基于结构和突变数据，我们认为ATP与PilT结合可诱导单个结构域的大规模运动，从而调节亚基之间的界面并促进协同运动。由于不受控制的回缩导致无毛状细胞，回缩的力量被与IV型预毛状蛋白具有相似n端序列的其他蛋白质抵消。本提案的三个具体目标旨在提高我们对毛收缩和平衡机制的理解。我们将同时采用自下而上和自上而下的方法，利用我们在蛋白质结构/功能分析方面的专业知识。具体而言，我们将(1)对铜绿假单胞菌和淋病奈瑟菌PilT结合的各种核苷酸类似物进行x射线晶体学和小角度散射研究，并对所得菌株进行基于结构的诱变和表型分析(2)通过在非毛状生物中表达确定毛状生物发生蛋白的最小集合(3)对低丰度的prepilin样蛋白进行蛋白质晶体学和定点诱变。这些实验的总体结果将更好地理解广泛存在的细菌毒力因子IV型菌毛收缩的分子机制。最终，这些知识将用于设计控制或阻断这一途径的方法，从而防止许多微生物疾病的传播。

DESCRIPTION (provided by applicant): Type IV pili are extracellular bacterial filaments required for attachment to surfaces and for pathogenesis in many plant, animal, and human infections (including bacterial meningitis caused by Neisseria meningitidis, gonorrhea caused by N. gonorrhoeae, and lung infections of cystic fibrosis patients caused by Pseudomonas aeruginosa). Through the work of the intracellular ATPase PilT, pili are retracted into the cell from which they emerge. Retraction leads to motility on surfaces and activates signaling pathways in eukaryotic target cells. Based on structural and mutagenesis data we suggest that ATP binding to PilT induces large-scale movements of individual domains, which adjusts the interface between subunits and promotes synergistic motor movements. Because unchecked retraction leads to non-piliated cells, the force of retraction is counterbalanced by other proteins with similar N-terminal sequences to Type IV prepilins. The three specific aims of this proposal are designed to improve our understanding of the mechanism of pilus retraction and counterbalance. We will take a simultaneous bottom-up & top-down approach that capitalizes on our expertise in protein structure/function analysis. Specifically, we will (1) carry out X-ray crystallography and small angle scattering studies of P. aeruginosa and N. gonorrhoeae PilT bound to various nucleotide analogues plus structure-based mutagenesis and phenotypic analysis of the resulting strains (2) identify the minimal set of pilus biogenesis proteins by expression in a non-piliated organism and (3) carry out protein crystallography and site directed mutagenesis of the low abundance prepilin-like proteins. The overall result of these experiments will be a better understanding of the molecular mechanism of retraction of the widespread bacterial virulence factor Type IV pili. Ultimately this knowledge will be used in designing approaches to control or block this pathway, thereby preventing the spread of many microbial diseases.