Structural Microbiology of Type IV Pilus Retraction

描述(申请人提供)：IV型菌毛是许多植物、动物和人类感染(包括脑膜炎奈瑟菌引起的细菌性脑膜炎、淋球菌引起的淋病和铜绿假单胞菌引起的囊性纤维化患者的肺部感染)附着表面和致病所需的细胞外细丝。通过细胞内ATPase PILT的工作，菌毛被收回到它们出现的细胞中。回缩导致表面的运动，并激活真核细胞中的信号通路。基于结构和突变数据，我们认为ATP与PILT结合可以诱导单个结构域的大规模运动，从而调节亚单位之间的界面，促进协同运动。因为无节制的回缩导致无毛细胞，回缩的力量被其他具有与IV型前粘连蛋白相似的N-末端序列的蛋白质所抵消。这项建议的三个具体目的是为了提高我们对毛发缩回和平衡机制的理解。我们将采取自下而上和自上而下的同步方法，充分利用我们在蛋白质结构/功能分析方面的专业知识。具体地说，我们将(1)进行与各种核苷酸类似物结合的铜绿假单胞菌和淋球菌的X射线结晶学和小角散射研究，以及对产生的菌株进行基于结构的突变和表型分析，(2)通过在非纤毛生物体中表达来确定最小的菌毛生物发生蛋白集，以及(3)对低丰度的前毛蛋白样蛋白进行蛋白质结晶学和定点突变。这些实验的总体结果将是更好地理解广泛存在的细菌毒力因子IV型菌毛收回的分子机制。最终，这些知识将被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.