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

CFA/I组件由四个功能/结构组件组成： 位于尖端的次要亚基并作为粘合剂组分起作用。CfaB聚合成 菌毛的柄，因此被称为主要菌毛。CfaA作为周质分子伴侣发挥作用 护送CfaB或CfaE到它们在细菌外膜的组装位点。CfaC，命名为 usher蛋白是形成皮利组装位点的完整外膜蛋白。 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。我们已经确定了CfaE的全长结构; CfaEad上的受体结合位点集中在一簇带正电荷的残基上 (R181，R182和R67）。为了证实该位点的作用，将R181、R182和R67分别克隆到了 每种突变为丙氨酸并且突变蛋白不能凝集人红细胞， 暗示由这三个残基锚定的口袋作为假定的受体结合 域确定结合周围单个残基在血凝中的作用 位点，我们进一步做了十二个涉及残基的突变，这些残基要么是不变的（完全不变的）， 保守的）或对5类ETEC菌毛粘附素具有亚类特异性。由于这种 分析，我们发现所有带正电荷的残基（R181，R182，R67）都是绝对的。 受体结合所需的，而周围的残基显示改变的相互作用 红细胞和几个显示歧视行为，无论是人类A型或牛 红细胞种类我们还确定了主要菌毛蛋白（CfaB）的亚基结构。为 我们首次阐明了ETEC主要菌毛蛋白亚基CfaB的原子结构， 定植因子抗原I（CFA/I）菌毛。这些数据用于构建模型， CFA/I菌毛的两种形态学形式都在体内观察到， 它通常被组装到其中，以及延伸的、未缠绕的构造。建模与 确证的突变数据表明，脯氨酸异构化参与了 CFA/I菌毛的螺旋和延伸形式之间的转换。我们的发现证实了 在5类菌毛（来自主要引起 胃肠道疾病）和1类皮利（来自引起泌尿、呼吸和 其它感染）。他们还建议 菌毛类型之间的形态学和生物化学差异，无论类别如何， 提供结构特化，促进每种细菌致病型在其 最好的微环境最后，我们提出了细菌使用的结构证据， 抗原变异，以逃避宿主免疫反应，在残基占据预测的 CfaB和相关5类菌毛蛋白表面暴露面显示出高得多的遗传序列 比菌毛蛋白的其余部分的可变性。

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.