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

CFA/I组件由四个功能/结构组件组成：CfaE是位于末端的次亚单位，具有粘附性成分的功能。CFAB聚合成菌毛的茎，因此被命名为主要菌毛素。CFAA起着周质伴侣的作用，护送CFAB或CFAE到它们在细菌外膜的组装位置。CfaC被命名为Usher蛋白，是形成菌毛组装部位的完整外膜蛋白。作为NIHS生物防御计划的一部分，我的实验室一直在研究ETEC的CFA/I结构。我们已经确定了CfaE的全长结构；在CfaEad上定位了一个以一簇带正电的残基(R181、R182和R67)为中心的可能的受体结合部位。为了证实这个位点的作用，R181、R182和R67分别突变为丙氨酸，突变的蛋白不能凝集人红细胞，这意味着这三个残基锚定的口袋可能是受体结合结构域。为了确定结合位点周围单个残基在血凝中的作用，我们进一步对5类ETEC菌毛粘附素进行了12个残基的突变，这些残基要么是不变的(完全保守)，要么是亚类特有的。结果，我们发现所有带正电荷的残基(R181，R182，R67)都是受体结合所必需的，而周围的残基与红细胞的相互作用发生了变化，其中几个残基对人类A型或牛红细胞物种表现出歧视行为。我们还确定了主要的Pilin(CFAB)的亚基结构。我们首次从定居因子抗原I(CFA/I)菌毛中阐明了ETEC主要菌毛蛋白亚基CFAB的原子结构。这些数据被用来构建两种形态的CFA/I菌毛的模型，这两种形态都是在体内观察到的，即它通常组装成的螺旋丝，以及延伸的、未缠绕的构象。模拟和确证突变数据表明，在CFA/I菌毛的螺旋形式和扩展形式之间的转换过程中涉及到脯氨酸异构化。我们的发现证实了5类菌毛(主要来自引起胃肠道疾病的细菌)和1类菌毛(来自引起泌尿、呼吸道和其他感染的细菌)之间在结构上的强烈相似性，而没有显著的初级序列相似性。他们还指出，菌毛类型之间的形态和生化差异，无论其类别如何，都提供了结构特化，促进了每种细菌致病类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，因为占据预测的CFAB表面暴露表面的残基和相关的5类毛孔蛋白显示出比毛孔蛋白其余部分更高的遗传序列变异性。

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.