Structural Microbiology of Type IV Pilus Retraction

IV 型菌毛回缩的结构微生物学

• 批准号: 7795831
• 负责人: Katrina T Forest
• 金额: $ 29.84万
• 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/7795831
• 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 型菌毛是附着在表面以及许多植物、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和由铜绿假单胞菌引起的囊性纤维化患者肺部感染）中发病所需的细胞外细菌丝。通过细胞内 ATP 酶 PilT 的作用，菌毛被缩回到产生它们的细胞中。回缩导致表面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，我们认为 ATP 与 PilT 结合会诱导单个结构域的大规模运动，从而调整亚基之间的界面并促进协同运动。由于不受控制的回缩会导致细胞不形成毛毛，因此回缩的力量会被具有与 IV 型前毛毛蛋白相似的 N 末端序列的其他蛋白质所抵消。该提案的三个具体目标旨在提高我们对菌毛回缩和平衡机制的理解。我们将同时采取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。具体来说，我们将 (1) 对与各种核苷酸类似物结合的铜绿假单胞菌和淋病奈瑟菌 PilT 进行 X 射线晶体学和小角散射研究，并对所得菌株进行基于结构的诱变和表型分析 (2) 通过在非绒毛生物体中表达来鉴定菌毛生物发生蛋白的最小集，以及 (3) 进行蛋白质晶体学和定点诱变 低丰度的前毛蛋白样蛋白。这些实验的总体结果将有助于更好地理解广泛传播的细菌毒力因子 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该途径的方法，从而防止许多微生物疾病的传播。