Molecular Basis of Localized Adherence in E. coli

大肠杆菌局部粘附的分子基础

• 批准号: 7540930
• 负责人: MICHAEL S DONNENBERG
• 金额: $ 27.46万
• 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 2010-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7540930
• 关键词: ATP phosphohydrolase; Adherence; Adhesions; Adhesives; Architecture; Bacterial Adhesins; Binding; Binding Sites; Biogenesis; Calorimetry; Cell membrane; Classification; Complex; Cytoplasmic Tail; Data; Dependence; Epitopes; Escherichia coli; Family; Fimbriae Proteins; Light; 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; fimbria; 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.

性状（由申请方提供）：肠致病性大肠杆菌（EPEC）的菌毛形成（BFP）是一种重要的毒力因子，是IV型菌毛大家族的成员。BFP是由一个复杂的11个组件的机器组装，并可能服务于粘合剂的功能。该提案旨在了解BFP生物发生机的结构和功能以及BFP和BFP蛋白在发病机制中的功能。为此，本提案有四个具体目标，包括：1.确定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生物发生机的内膜子组件的这三（3）个关键组件之间的相互作用。所提出的实验将确定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Alkalin跨膜转运的假说。迄今为止的研究中出现了一个令人兴奋的假设， BfpU将Escherlin的疏水性氨基末端加帽，以允许后者从细胞质膜提取并运送到生长的菌毛。这一假设的各个方面将得到检验 通过检测BfpU对Bfplin分配到膜囊泡中的影响，并通过研究BFP生物发生机的周质结构域与BfpU和BfpU-Bfplin复合物之间的相互作用。3.定义BfpB的拓扑和绑定交互。外膜分泌素蛋白BfpB的拓扑结构将通过系统插入来定义 表位和蛋白酶切割位点的分析，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.通过对BFP结构的研究，推测BFP的结构及其作为粘合剂的作用。关于α 1-β-glycolin单体的结构和诱变数据将用于对菌毛本身进行建模。进一步的研究将调查磷脂的结合，并确定受体结合 蛋白质上的位点。将解决的远亲B6变异体的结构，揭示序列变异的菌毛结构和免疫原性的作用。

项目成果

The Cpx envelope stress response both facilitates and inhibits elaboration of the enteropathogenic Escherichia coli bundle-forming pilus.

• DOI: 10.1111/j.1365-2958.2010.07145.x
• 发表时间: 2010-06-01
• 期刊: Molecular microbiology
• 影响因子: 3.6
• 作者: Vogt SL;Nevesinjac AZ;Humphries RM;Donnenberg MS;Armstrong GD;Raivio TL
• 通讯作者: Raivio TL

BfpL is essential for type IV bundle-forming pilus biogenesis and interacts with the periplasmic face of BfpC.

BfpL 对于 IV 型束形成菌毛生物发生至关重要，并与 BfpC 的周质面相互作用。

• DOI: 10.1099/mic.0.060889-0
• 发表时间: 2012
• 期刊: Microbiology (Reading, England)
• 作者: DeMasi,Leon;Szmacinski,Henryk;Schreiber,Wiebke;Donnenberg,MichaelS
• 通讯作者: Donnenberg,MichaelS

From alpha to beta: identification of amino acids required for the N-acetyllactosamine-specific lectin-like activity of bundlin.

• DOI: 10.1111/j.1365-2958.2009.06679.x
• 发表时间: 2009-05
• 期刊: Molecular microbiology
• 影响因子: 3.6
• 作者: Humphries RM;Donnenberg MS;Strecker J;Kitova E;Klassen JS;Cui L;Griener TP;Mulvey GL;Armstrong GD
• 通讯作者: Armstrong GD

Complete resonance assignments of bundlin (BfpA) from the bundle-forming pilus of enteropathogenic Escherichia coli.

来自肠致病性大肠杆菌的束形成菌毛的束蛋白 (BfpA) 的完整共振分配。

• DOI: 10.1023/b:jnmr.0000032511.89525.64
• 发表时间: 2004
• 期刊: Journal of biomolecular NMR
• 影响因子: 2.7
• 作者: Ramboarina,Stéphanie;Fernandes,Paula;Simpson,Peter;Frankel,Gad;Donnenberg,Michael;Matthews,Stephen
• 通讯作者: Matthews,Stephen

N-acetyllactosamine-induced retraction of bundle-forming pili regulates virulence-associated gene expression in enteropathogenic Escherichia coli.

• DOI: 10.1111/j.1365-2958.2010.07192.x
• 发表时间: 2010-06-01
• 期刊: Molecular microbiology
• 影响因子: 3.6
• 作者: Humphries RM;Griener TP;Vogt SL;Mulvey GL;Raivio T;Donnenberg MS;Kitov PI;Surette M;Armstrong GD
• 通讯作者: Armstrong GD