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

基本信息

批准号：
9556346
负责人：
di s xia
金额：
$ 6.76万
依托单位：
DIVISION OF BASIC SCIENCES - NCI
依托单位国家：
美国
项目类别：
财政年份：
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/9556346
关键词：
Adherence Adhesives Alanine Alveolar Amino Acids Antigenic Variation Antigens Bacteria Bacterial Adhesins Behavior Binding Binding Sites Biochemical Biogenesis C-terminal Cattle Cell surface Cells Charge Child Choline Complement Complex Consensus Consensus Sequence Country Crystallization Data Diarrhea Distal Docking Domestic Animals Electrolytes Engineering Enterotoxins Epithelial Cells Erythrocytes Escherichia coli Face Family Fiber Filament Fimbriae Proteins Fimbrial Adhesins Galactose Galactosylceramides Gastrointestinal Diseases Genetic Geometry Glycosphingolipids Hemagglutination Human I-antigen Immune response In Vitro Individual Infant Mortality Infection Intestines Kinetics Lecithin Length Ligands Link Liquid substance Location Lung Mammalian Cell Mediating Membrane Mental Depression Minor Modeling Molecular Molecular Chaperones Molecular Conformation Morphology Mutagenesis Mutate Mutation Mutation Analysis N-terminal Neighborhoods Pathogenesis Pathogenicity Pathway interactions Periodicity Phenotype Phospholipids Phosphorylcholine Pilum Plague Plasmids Polymers Proline Proteins Recombinants Research Resolution Resources Role Rotavirus Shapes Site Site-Directed Mutagenesis Structure Surface Surface Antigens Time Traveler&apos s diarrhea Usher Proteins Variant Work Yersinia pestis base beta pleated sheet colonization factor antigens driving force enterotoxigenic Escherichia coli fimbria flexibility in vivo inhibitor/antagonist insight macromolecule macrophage microbial mutant pathogen periplasm polymerization prevent protein complex receptor receptor binding reconstruction respiratory retinal rods urinary

项目摘要

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. 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. We elucidated, for the first time, 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. More recently, we have also determined the crystal structure of CfaA, the chaperone component that is essential for assembly of CFA/I fimbriae. Pili assembled by the chaperone-usher pathway (CUP) require periplasmic chaperones that assist subunit folding, maintain their stability, and escort them to the site of bioassembly. Until now, CUP chaperones have been classified into two families, FGS and FGL, based on the short and long length of the subunit-interacting loops between its F1 and G1 beta-strands, respectively. CfaA is the chaperone for assembly of colonization factor antigen I (CFA/I) pili of enterotoxigenic E. coli (ETEC), a cause of diarrhea in travelers and young children. Here, the crystal structure of CfaA along with mutational analyses reveals some unique structural and functional features, leading us to propose a separate family for CfaA and closely related chaperones. Phenotypic changes resulting from mutations in regions unique to this chaperone family provides insight into their function. Moreover, those regions that distinguish CfaA from the FGL and FGS chaperone families appear to influence interactions with their cognate subunits and usher proteins during pilus assembly. Despite the lack of detailed understanding with respect to the energetics of fimbria assembly, periplasmic chaperones are thought to serve both as kinetic and energy traps by forming binary complexes with pilin subunits during subunit refolding, which prevents subunits from aggregating and subsequently drives their polymerization into ordered helical assemblies. The assembly of CFA/I fimbriae, an archetype in the family of class 5 fimbriae from enterotoxigenic E. coli, is assisted by the periplasmic chaperone CfaA, which belongs to a newly classified group of periplasmic chaperones. In this work, we show that unlike pilin subunits of other bacterial fimbriae, CfaB, the major pilin subunits of CFA/I, are stable in the absence of the chaperone and are able to spontaneously refold and polymerize into cyclic trimers both in vivo and in vitro. We further show that chaperone CfaA is able to kinetically trap CfaB to form a metastable complex. Stabilization of the CfaA and CfaB heterodimer by mutations led to the crystal structure of the complex, revealing distinctive interactions between CfaA and CfaB through donor-strand complementation and cleft-mediated anchorage. Mutagenesis indicated that donor-strand complementation controls the stability of the chaperone-subunit complex and the cleft-mediated anchorage of the subunit C-terminus appears to assist in subunit refolding. Surprisingly, over-stabilization of the chaperon-subunit complex led to delayed fimbria assembly, whereas destabilizing the complex resulted in no fimbriation. Thus, CfaA functions predominantly as a kinetic trap fine-tuned to avoid off-pathway subunit self-polymerization, which results in energetically favorable trimers and could serve as a driving force for CFA/I pilus assembly, representing an energetic landscape unique to class 5 fimbria assembly.

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。 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。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。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。由分子伴侣引导途径 (CUP) 组装的菌毛需要周质分子伴侣来协助亚基折叠、维持其稳定性并护送它们到达生物组装位点。到目前为止，CUP 伴侣分子根据其 F1 和 G1 β 链之间的亚基相互作用环的长度分别分为 FGS 和 FGL 两个家族。 CfaA 是产肠毒素大肠杆菌 (ETEC) 定植因子抗原 I (CFA/I) 菌毛组装的伴侣，ETEC 是旅行者和幼儿腹泻的原因。在这里，CfaA 的晶体结构以及突变分析揭示了一些独特的结构和功能特征，使我们为 CfaA 和密切相关的伴侣提出了一个单独的家族。该伴侣家族独特区域的突变导致的表型变化提供了对其功能的深入了解。此外，那些将 CfaA 与 FGL 和 FGS 伴侣家族区分开来的区域似乎会影响菌毛组装过程中与其同源亚基和引导蛋白的相互作用。尽管对菌毛组装的能量学缺乏详细的了解，但周质伴侣被认为通过在亚基重折叠过程中与菌毛蛋白亚基形成二元复合物来充当动能陷阱和能量陷阱，从而防止亚基聚集并随后驱动它们聚合成有序的。螺旋组件。 CFA/I 菌毛是产肠毒素大肠杆菌第 5 类菌毛家族的原型，它的组装受到周质伴侣 CfaA 的协助，CfaA 属于新分类的周质伴侣。在这项工作中，我们表明，与其他细菌菌毛的菌毛蛋白亚基不同，CfaB（CFA/I 的主要菌毛蛋白亚基）在没有伴侣的情况下是稳定的，并且能够在体内和体外自发地重新折叠并聚合成环状三聚体。体外。我们进一步表明伴侣 CfaA 能够动力学捕获 CfaB 形成亚稳态复合物。通过突变使 CfaA 和 CfaB 异二聚体稳定，形成了复合物的晶体结构，揭示了 CfaA 和 CfaB 通过供体链互补和裂口介导的锚定之间独特的相互作用。诱变表明，供体链互补控制伴侣-亚基复合物的稳定性，并且亚基 C 末端的裂口介导的锚定似乎有助于亚基重折叠。令人惊讶的是，伴侣-亚基复合物的过度稳定导致菌毛组装延迟，而破坏复合物的稳定则不会导致菌毛组装。因此，CfaA 主要作为一个动态陷阱进行微调，以避免偏离路径的亚基自聚合，从而产生能量上有利的三聚体，并可以作为 CFA/I 菌毛组装的驱动力，代表 5 类独有的能量景观菌毛组装。

项目成果

期刊论文数量（4）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Preliminary X-ray diffraction analysis of CfaA, a molecular chaperone essential for the assembly of CFA/I fimbriae of human enterotoxigenic Escherichia coli.

CfaA 的初步 X 射线衍射分析，CfaA 是人产肠毒素大肠杆菌 CFA/I 菌毛组装所必需的分子伴侣。

DOI：
10.1107/s2053230x13033967
发表时间：
2014
期刊：
Acta crystallographica. Section F, Structural biology communications
影响因子：
0
作者：
Bao,Rui;Esser,Lothar;Poole,Steven;McVeigh,Annette;Chen,YuXing;Savarino,StephenJ;Xia,Di
通讯作者：
Xia,Di

Structure of CfaA suggests a new family of chaperones essential for assembly of class 5 fimbriae.