Molecular Basis of Localized Adherence in E. coli

大肠杆菌局部粘附的分子基础

• 批准号: 7163804
• 负责人: MICHAEL S DONNENBERG
• 金额: $ 28.16万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-04-01 至 2009-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7163804
• 关键词: ATP phosphohydrolase; Adherence; Adhesions; Adhesives; Architecture; Bacterial Adhesins; Binding; Binding Sites; Biogenesis; Calorimetry; Cell membrane; Classification; Complex; Cytoplasmic Tail; Data; Dependence; Endopeptidases; Epitopes; Escherichia coli; Family; Fimbria of hippocampus; Fimbriae Proteins; Light; Localized; Membrane; Modeling; Molecular; Molecular Conformation; Mutagenesis; Nuclear Magnetic Resonance; Pathogenesis; Peptide Hydrolases; Phospholipids; Pilum; Proteins; Role; Secretin; Site; Solutions; Spectrometry; Structure; Surface; Techniques; Testing; Time; Titrations; Variant; Vesicle; Virulence Factors; base; enteropathogenic Escherichia coli; immunogenicity; member; monomer; periplasm; receptor binding; research study; yeast two hybrid system

DESCRIPTION (provided by applicant): The bundle-forming pilus (BFP) of enteropathogenic Escherichia coli (EPEC) is an important virulence factor and a member of the large type IV fimbria family. BFP are assembled by a complex 11-component machine and may serve an adhesive function. This proposal seeks an understanding of the architecture and function of the BFP biogenesis machine and of the function of BFP and bundlin in pathogenesis. Toward these ends, this proposal has four specific aims involving: 1. To define the structural and functional interactions among BfpC, BfpD, and BfpE. Prior experiments have established interactions among these three (3) critical components of the inner membrane subassembly of the BFP biogenesis machine. The proposed experiments will define binding sites for BfpD and BfpE in BfpC, and for BfpE and BfpC in BfpD and will determine the effect of BfpC and BfpE binding on the conformation of BfpD. 2. To explore the hypothesis that BfpU ferries bundlin across the cytoplasmic membrane. An exciting hypothesis that has emerged from studies to date states that BfpU caps the hydrophobic amino terminus of bundlin to allow the latter to be extracted from the cytoplasmic membrane and ferried to the growing pilus. Aspects of this hypothesis will be tested by examining the effect of BfpU on bundlin partitioning into membrane vesicles and by investigating interactions between periplasmic domains of the BFP biogenesis machine and both BfpU and a BfpU-bundlin complex. 3. To define the topology and binding interactions of BfpB. The topology of the outer membrane secretin protein BfpB will be defined by systematic insertion of epitopes and protease cleavage sites into the protein to determine which domains are surfaced-exposed. The binding sites for BfpG and BfpB in BfpB will be identified. 4. To deduce the structure of BFP and its function as an adhesion through studies of bundlin. Structural and mutagenesis data on the a1-bundlin monomer will be used to model the pilus itself. Further studies will investigate the binding of phospholipids to bundlin and determine the receptor binding sites on the protein. The structure of the distantly related B6 variant of bundlin will be solved to shed light on the role of sequence variation in pilus structure and immunogenicity.

描述（由申请人提供）：致病性大肠杆菌（EPEC）的成束菌毛（BFP）是一种重要的毒力因子，并且是大IV型菌毛家族的成员。 BFP 由复杂的 11 部件机器组装而成，可起到粘合功能。该提案旨在了解 BFP 生物发生机的架构和功能以及 BFP 和 Bundlin 在发病机制中的功能。为了实现这些目标，该提案有四个具体目标，包括： 1. 定义 BfpC、BfpD 和 BfpE 之间的结构和功能相互作用。先前的实验已经建立了 BFP 生物发生机内膜组件的这三 (3) 个关键组件之间的相互作用。拟议的实验将定义 BfpC 中 BfpD 和 BfpE 的结合位点，以及 BfpD 中 BfpE 和 BfpC 的结合位点，并将确定 BfpC 和 BfpE 结合对 BfpD 构象的影响。 2. 探讨BfpU介导bundlin穿过细胞质膜的假说。迄今为止的研究中出现的一个令人兴奋的假设表明 BfpU 给束蛋白的疏水性氨基末端加帽，以允许后者从细胞质膜中提取并运送到正在生长的菌毛。该假设的各个方面将得到检验 通过检查 BfpU 对 Bundlin 分配到膜囊泡的影响，并研究 BFP 生物发生机的周质结构域与 BfpU 和 BfpU-bundlin 复合物之间的相互作用。 3. 定义BfpB的拓扑和绑定交互。外膜促胰液素蛋白 BfpB 的拓扑结构将通过系统插入来定义 将表位和蛋白酶切割位点分析到蛋白质中，以确定哪些结构域是表面暴露的。将鉴定 BfpB 中 BfpG 和 BfpB 的结合位点。 4.通过bundlin的研究推导BFP的结构及其作为粘附剂的功能。 a1-bundlin 单体的结构和诱变数据将用于模拟菌毛本身。进一步的研究将调查磷脂与 Bundlin 的结合并确定受体结合 蛋白质上的位点。远缘相关的 Bundlin B6 变体的结构将得到解决，以阐明序列变异在菌毛结构和免疫原性中的作用。