Structural studies of fimbriae of enterotoxigenic E. coli (ETEC)

产肠毒素大肠杆菌 (ETEC) 菌毛的结构研究

• 批准号: 7965581
• 负责人: di s xia
• 金额: $ 10.61万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7965581
• 关键词: Acute; Adherence; Adhesives; Alanine; Antigenic Variation; Architecture; Bacteria; Bacterial Pili; Behavior; Binding Sites; Biochemical; Cattle; Charge; Chemical Interference; Child; Country; Data; Diarrhea; Distal; Electrolytes; Enterobacteriaceae; Enterotoxins; Erythrocytes; Face; Filament; Fimbriae Proteins; Fimbrial Adhesins; Gastrointestinal Diseases; Genetic; Hemagglutination; Human; Immune response; Individual; Infection; Intestines; Knowledge; Length; Liquid substance; Membrane; Membrane Proteins; Minor; Modeling; Molecular Chaperones; Molecular Conformation; Mutate; Mutation; Names; Pathway interactions; Pilum; Proline; Proteins; Resolution; Resources; Role; Site; Small Intestines; Structural Biologist; Structure; Surface; Time; Traveler' s diarrhea; Vaccines; Work; biodefense; colonization factor antigens; enterotoxigenic Escherichia coli; fimbria; in vivo; interest; mutant; periplasm; programs; receptor; receptor binding; respiratory; retinal rods; urinary

The CFA/I assembly consists of four functional/structural components: CfaE is a tip-located minor subunit and functions as an adhesive component. CfaB polymerizes into the stalk of a pilus and is therefore named major pilin. CfaA functions as a periplasmic chaperone escorting CfaB or CfaE to their assembly site at the bacterial outer membrane. CfaC, named the usher protein, is an integral outer membrane protein forming the assembly site for the pili. As part of the NIHs Biodefense program, my lab has been working on structure determination of CFA/I of ETEC. We have determined the full-length structure of CfaE; a putative receptor-binding site on the CfaEad centered on a cluster of positively charged residues (R181, R182 and R67) was located. To confirm the role of this site, R181, R182, and R67 were each mutated to alanines and the mutant proteins failed to agglutinate human erythrocytes, implicating the pocket anchored by these three residues as the putative receptor-binding domain. To determine the role in hemagglutination of individual residues surround the binding site, we further made twelve mutations involving residues that are either invariant (fully conserved) or subclass-specific for the Class 5 ETEC fimbrial adhesins. As a result of this analysis, we found that all positively charged residues (R181, R182, R67) are absolutely required for receptor binding, whereas those surrounding residues display altered interactions with red-blood cells and several show discriminatory behavior to either human type-A or bovine red cell species. We have also determined subunit structures of the major pilin (CfaB). For the first time, we elucidate atomic structures of an ETEC major pilin subunit, CfaB from colonization factor antigen I (CFA/I) fimbriae. These data are used to construct models for two morphological forms of CFA/I fimbriae that are both observed in vivo, the helical filament into which it is typically assembled, and an extended, unwound conformation. Modeling and corroborative mutational data indicate that proline isomerization is involved in the conversion between the helical and extended forms of CFA/I fimbriae. Our findings affirm the strong structural similarities seen between Class 5 fimbriae (from bacteria primarily causing gastrointestinal disease) and Class 1 pili (from bacteria that cause urinary, respiratory and other infections) in the absence of significant primary sequence similarity. They also suggest that morphological and biochemical differences between fimbrial types, regardless of class, provide structural specialization that facilitates survival of each bacterial pathotype in its preferred host microenvironment. Lastly, we present structural evidence for bacterial use of antigenic variation to evade host immune responses, in that residues occupying the predicted surface-exposed face of CfaB and related Class 5 pilins show much higher genetic sequence variability than the remainder of the pilin protein.

CFA/I 组件由四个功能/结构组件组成：CfaE 是位于尖端的次要亚基，用作粘合组件。 CfaB 聚合成菌毛茎，因此被命名为大菌毛蛋白。 CfaA 作为周质伴侣，护送 CfaB 或 CfaE 到达细菌外膜的组装位点。 CfaC，称为引导蛋白，是一种完整的外膜蛋白，形成菌毛的组装位点。 作为 NIH 生物防御计划的一部分，我的实验室一直致力于 ETEC 的 CFA/I 结构测定。我们已经确定了CfaE的全长结构；定位了 CfaEad 上以一组带正电残基（R181、R182 和 R67）为中心的假定受体结合位点。为了证实该位点的作用，R181、R182和R67分别突变为丙氨酸，并且突变蛋白无法凝集人红细胞，这表明这三个残基锚定的口袋是假定的受体结合结构域。为了确定结合位点周围单个残基在血凝中的作用，我们进一步进行了 12 个突变，涉及 5 类 ETEC 菌毛粘附素不变（完全保守）或亚类特异性的残基。作为该分析的结果，我们发现所有带正电荷的残基（R181、R182、R67）对于受体结合绝对是必需的，而周围的残基显示出与红细胞的相互作用发生改变，并且有几个残基表现出对任一人类类型的歧视行为- A 或牛红细胞种类。我们还确定了主要菌毛蛋白 (CfaB) 的亚基结构。我们首次从定植因子抗原 I (CFA/I) 菌毛中阐明了 ETEC 主要菌毛蛋白亚基 CfaB 的原子结构。这些数据用于构建 CFA/I 菌毛的两种形态的模型，这两种形态都在体内观察到，即通常组装成的螺旋丝，以及延伸的未缠绕构象。建模和确凿的突变数据表明脯氨酸异构化参与了 CFA/I 菌毛的螺旋形式和延伸形式之间的转换。我们的研究结果证实了第 5 类菌毛（来自主要引起胃肠道疾病的细菌）和第 1 类菌毛（来自引起泌尿、呼吸道和其他感染的细菌）之间存在很强的结构相似性，但缺乏显着的一级序列相似性。他们还表明，无论类别如何，菌毛类型之间的形态和生化差异提供了结构特化，有利于每种细菌致病型在其首选宿主微环境中的生存。最后，我们提出了细菌利用抗原变异来逃避宿主免疫反应的结构证据，因为占据 CfaB 和相关 5 类菌毛蛋白的预测表面暴露面的残基显示出比菌毛蛋白其余部分更高的遗传序列变异性。