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）：肠病毒性大肠杆菌（EPEC）的束形成菌毛（BFP）是重要的毒力因素，也是大型IV纤维菌家族的成员。 BFP由复杂的11组分机组装，可以发挥粘合功能。该建议寻求了解BFP生物发生机的结构和功能以及BFP和Bundlin在发病机理中的功能。在这些目的方面，该建议具有四个涉及的特定目标：1。定义BFPC，BFPD和BFPE之间的结构和功能相互作用。先前的实验已经在BFP生物发生机的这三（3）个临界组件之间建立了相互作用。提出的实验将在BFPC中定义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。探讨以下假设：BFPU渡轮在整个细胞质膜上。从研究到迄今为止的一个令人兴奋的假设指出 BFPU限制了Bundlin的疏水氨基末端，使后者可以从细胞质膜中提取并拧紧到生长的菌毛中。该假设的各个方面将进行检验 通过检查BFPU对BUNDLIN分配到膜囊泡中的影响，并研究BFP生物发生机和BFPU和BFPU-Bundlin复合物之间的张质质域之间的相互作用。 3。定义BFPB的拓扑和结合相互作用。外膜分泌蛋白BFPB的拓扑结构将由系统插入定义 表位和蛋白酶裂解位点进入蛋白质，以确定哪些结构域暴露在外。 BFPB中BFPG和BFPB的结合位点将被识别。 4。通过对邦德林的研究来推断BFP的结构及其功能作为粘附。 A1-Bundlin单体上的结构和诱变数据将用于对PILUS本身进行建模。进一步的研究将研究磷脂与Bundlin的结合并确定受体结合 蛋白质上的位置。遥远相关的邦德林B6变体的结构将求解，以阐明序列变化在壁pil结构和免疫原性中的作用。