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: 8349127
• 负责人: di s xia
• 金额: $ 11.29万
• 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/8349127
• 关键词: Adherence; Adhesives; Alanine; Amino Acids; Antigenic Variation; Bacteria; Bacterial Adhesins; Behavior; Binding; Binding Sites; Biochemical; Biogenesis; C-terminal; Cattle; Cell surface; Cells; Charge; Complement; Complex; Country; Data; Diarrhea; Disease Outbreaks; Distal; Docking; Domestic Animals; Electrolytes; Engineering; Enterotoxins; Erythrocytes; Escherichia coli; Face; Fiber; Filament; Fimbriae Proteins; Fimbrial Adhesins; Genetic; Hemagglutination; Human; Immune response; Individual; Infant Mortality; Intestines; Isomerase; Knowledge; Ligands; Liquid substance; Location; Maps; Mediating; Membrane; Mental Depression; Minor; Modeling; Molecular Chaperones; Molecular Conformation; Morphology; Mutate; Mutation; N-terminal; Neighborhoods; Operon; Pathogenesis; Pathway interactions; Pilum; Plasmids; Process; Proline; Proteins; Recombinants; Resources; Role; Rotavirus; Sequence Analysis; Shapes; Site; Structure; Surface; Surface Antigens; System; Therapeutic; Traveler' s diarrhea; Vaccines; Variant; analog; base; beta pleated sheet; colonization factor antigens; enterotoxigenic Escherichia coli; fimbria; flexibility; macromolecule; membrane assembly; microbial; mutant; pathogen; periplasm; protein complex; receptor; receptor binding; reconstruction; retinal rods; small molecule; vaccine development

A variant of CfaE, donor strand complemented CfaE (dscCfaE), containing a C-terminal hairpin linker followed by the first 19 amino acid residues derived from the N-terminus of the major fimbrial subunit CfaB was purified to homogeneity. The dscCfaE protein was readily crystallized and the structure was determined. The dscCfaE molecule consists of two domains of roughly equal size. The N-terminal domain of CfaE is referred to as the adhesin domain (CfaEad) and is represented in the structure from residues A23 to D200. It consists of one anti-parallel beta-sheet (Sheet 1) and one mixed beta-sheet (Sheet 2). The C-terminal domain immediately follows the short three-residue linker (K201-G202-N203) and mediates attachment of the adhesive subunit to the main body of the fimbria. It is therefore termed the pilin domain (CfaEpd). The pilin domain folds into a beta-sandwich with a topology reminiscent of the adhesin domain. Both CfaEad and CfaEpd beta-structures display a topology that resembles the v-type Ig fold with nine beta-strands. In order to understand how the major and minor subunits are assembled into a CFA/I fimbria, we further engineered the donor strand complemented CfaEB complex (dscCfaEB) construct, which was expressed in E. coli. The recombinant dscCfaEB protein was purified and crystallized. The crystal structure of the CfaEB complex was determined, providing structural information on not only the major subunit CfaB, but also the geometry of the connection between the major and minor subunit. In addition to the CfaEB complex, we also determined crystal structures for the major-major subunit complexes CfaBB and CfaBBB, providing a basis for constructing a model of CFA/I pilus consistent with EM reconstructions of purified CFA/I pilus. EM studies of ETEC strains observed two-distinct forms of CFA/I pili, a helical filament 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. This also suggested the existence of a possible prolinyl isomerase activity for CfaC. Our findings affirm the strong structural similarities seen between Class 5 fimbriae and Class 1 pili 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. Our model supports the notion that bacteria use antigenic variation to evade host immune response 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. Located at the upper surface of CfaEad distal to the CfaEpd, R181, which was previously known to be important for binding, is found in a positively charged depression and surrounded by a cluster of residues that are highly conserved in the Class 5 fimbrial adhesins, including residues from three different loops (i.e., B'-C, D'-E, and F-G loops). This pocket thus appears to be a suitable location to which a negatively charged sialylated receptor might bind. To confirm the role of this domain, R67, which is adjacent to R181, was mutated to alanine (dscCfaE/R67A) and purified. Bead-adsorbed dscCfaE/R67A failed to agglutinate human erythrocytes, similar to our previous findings for the dscCfaE/R181A mutant. These results implicate the pocket anchored by these two residues as the putative receptor-binding domain. To determine the role in hemagglutination of individual residues in the neighborhood of R181, we introduced site-specific mutations into CfaE in the plasmid pMAM2, which encodes all components of the CFA/I and directs surface expression of mutant fimbriae with single site mutations of CfaE. Twelve such mutations involving residues that are either invariant (fully conserved) or are subclass-specific for Class 5 ETEC fimbrial adhesins were introduced. All positively charged residues (R181, R182, R67) are absolutely required for receptor binding and cluster together to form a positively charged center. The positively charged center of the binding pocket is surrounded by a band of subclass-specific residues. Mutations of those residues display altered interactions with red cells and several show discriminatory behavior to either human type-A or bovine red cell species. Recently, we have also determined the crystal structure of CfaA, the chaperone component that is essential for assembly of CFA/I fimbriae.

CfaE 的变体，供体链互补的 CfaE (dscCfaE)，含有 C 端发夹接头，后接源自主要菌毛亚基 CfaB N 端的前 19 个氨基酸残基，纯化至同质。 dscCfaE 蛋白很容易结晶并确定其结构。 dscCfaE 分子由两个大小大致相等的结构域组成。 CfaE 的 N 端结构域称为粘附素结构域 (CfaEad)，在残基 A23 至 D200 的结构中表示。它由一个反平行 β 折叠（表 1）和一个混合 β 折叠（表 2）组成。 C 端结构域紧​​接在短的三残基接头 (K201-G202-N203) 后面，并介导粘附亚基与菌毛主体的附着。因此它被称为菌毛蛋白结构域 (CfaEpd)。菌毛蛋白结构域折叠成β-三明治，其拓扑结构让人想起粘附素结构域。 CfaEad 和 CfaEpd β 结构都显示出类似于具有 9 个 β 链的 v 型 Ig 折叠的拓扑结构。为了了解主要和次要亚基如何组装成 CFA/I 菌毛，我们进一步设计了供体链互补的 CfaEB 复合物 (dscCfaEB) 构建体，该构建体在大肠杆菌中表达。重组dscCfaEB蛋白被纯化并结晶。确定了 CfaEB 复合物的晶体结构，不仅提供了主要亚基 CfaB 的结构信息，还提供了主要亚基和次要亚基之间连接的几何形状。除了CfaEB复合物外，我们还确定了主要亚基复合物CfaBB和CfaBBB的晶体结构，为构建与纯化CFA/I菌毛的电镜重建一致的CFA/I菌毛模型提供了基础。 ETEC 菌株的电镜研究观察到两种不同形式的 CFA/I 菌毛：螺旋丝和延伸的未缠绕构象。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。这也表明 CfaC 可能存在脯氨酰异构酶活性。我们的研究结果证实，在缺乏显着的一级序列相似性的情况下，第 5 类菌毛和第 1 类菌毛之间存在很强的结构相似性。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。我们的模型支持细菌利用抗原变异来逃避宿主免疫反应的观点，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高得多的遗传序列变异性。 R181 位于 CfaEad 远端 CfaEpd 的上表面，之前已知对结合很重要，它被发现位于带正电荷的凹陷中，并被一组在 5 类菌毛粘附素中高度保守的残基包围，包括来自三个不同环（即 B'-C、D'-E 和 F-G 环）的残基。因此，这个口袋似乎是带负电荷的唾液酸化受体可能结合的合适位置。为了确认该结构域的作用，将与 R181 相邻的 R67 突变为丙氨酸 (dscCfaE/R67A) 并进行纯化。珠吸附的 dscCfaE/R67A 未能凝集人红细胞，这与我们之前对 dscCfaE/R181A 突变体的发现类似。这些结果表明这两个残基锚定的口袋是假定的受体结合结构域。 为了确定 R181 附近各个残基在血凝中的作用，我们将位点特异性突变引入质粒 pMAM2 中的 CfaE，该质粒编码 CFA/I 的所有组件，并指导具有 CfaE 单位点突变的突变菌毛的表面表达。引入了 12 个涉及 5 类 ETEC 菌毛粘附素不变（完全保守）或亚类特异性残基的突变。所有带正电荷的残基（R181、R182、R67）都是受体结合所必需的，并聚集在一起形成带正电荷的中心。结合袋带正电荷的中心被亚类特异性残基带包围。这些残基的突变显示出与红细胞相互作用的改变，并且一些残基显示出对人类 A 型或牛红细胞物种的歧视行为。 最近，我们还确定了 CfaA 的晶体结构，CfaA 是 CFA/I 菌毛组装所必需的伴侣成分。