Structural Microbiology of Type IV Pilus Retraction

IV 型菌毛回缩的结构微生物学

• 批准号: 7629077
• 负责人: Katrina T Forest
• 金额: $ 29.3万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-06-01 至 2012-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7629077
• 关键词: ATP phosphohydrolase; Active Sites; Adherence; Adopted; Animals; Bacterial Meningitis; Binding; Biochemical; Biogenesis; Biological; Cell surface; Cells; Complement; Crystallography; Data; Engineering; Equilibrium; Escherichia coli; Filament; Fimbriae Proteins; Gonorrhea; Human; Hydrolysis; Individual; Infection; Knowledge; Length; Ligand Binding; Lung; Measurement; Mediating; Membrane; Microbiology; Modeling; Molecular; Molecular Conformation; Motion; Motor; Movement; Mutagenesis; N-terminal; Neisseria gonorrhoeae; Neisseria meningitidis; Nucleotides; Optics; Organism; Pathogenesis; Pathway interactions; Peptide Hydrolases; Pilum; Plants; Principal Investigator; Propilin; Proteins; Pseudomonas aeruginosa; Resolution; Roentgen Rays; Role; Rotation; Signal Pathway; Site-Directed Mutagenesis; Solutions; Structure; Surface; System; Testing; Virulence Factors; Work; X-Ray Crystallography; base; cell motility; cystic fibrosis patients; design; extracellular; forest; improved; in vivo; microbial disease; monomer; nucleotide analog; pathogen; prevent; programs; protein expression; protein function; protein structure; protein structure function; research study

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.

描述（由申请人提供）：IV型PILI是附着在表面上所需的细胞外细菌细胞和许多植物，动物和人类感染的发病机理（包括由脑膜炎脑膜炎引起的细菌性脑膜炎，淋病引起的淋病，由N. gonorrhoeae引起，由N. gonorrhoeae引起，由Cystic Fibrudos引起的垂体病引起。通过细胞内ATPase PILT的工作，Pili缩回到它们出现的细胞中。缩回导致表面上的运动，并激活真核靶细胞中的信号通路。基于结构和诱变数据，我们建议ATP与PILT结合会诱导单个域的大规模运动，从而调整亚基之间的界面并促进协同运动运动。由于未检查的缩回导致非细胞细胞，因此与IV型前蛋白具有类似的N末端序列的其他蛋白质缩回的力平衡。该提案的三个特定目的旨在提高我们对Pilus缩回和平衡机制的理解。我们将采用同时自下而上和自上而下的方法，该方法利用了我们在蛋白质结构/功能分析方面的专业知识。具体而言，我们将（1）对铜绿假单胞菌和蜂窝h。低丰度前蛋白样蛋白。这些实验的总体结果将是对广泛的细菌毒力因子IV型pili的缩回的分子机理的更好理解。最终，这些知识将用于设计或阻止此途径的方法，从而阻止许多微生物疾病的传播。