ABC transporter-mediated secretion of capsular polysaccharides

ABC 转运蛋白介导的荚膜多糖分泌

基本信息

批准号：
10412117
负责人：
Jochen Zimmer
金额：
$ 19.71万
依托单位：
UNIVERSITY OF VIRGINIA
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-06-01 至 2024-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10412117
关键词：
3-Dimensional ATP Hydrolysis ATP-Binding Cassette Transporters Acinetobacter Address Adhesions Anabolism Antibiotics Bacteria Biocompatible Materials Biological Models CRISPR/Cas technology Carbohydrates Cell Adhesion Cell Wall Cell membrane Cell surface Cells Complement Complex Cryoelectron Microscopy Cytosol Data Deposition Detection Detergents Development Diffusion Digestion Engineering Enterobacteriaceae Escherichia coli Excision Extracellular Structure Fluorescent Probes Foundations Future Genes Homologous Gene Hyaluronan Hydration status Immune Immune response Immunologics Innate Immune Response Laboratories Link Lipid Bilayers Lipids Mechanics Mediating Membrane Membrane Transport Proteins Microbial Biofilms Molecular Molecular Probes Molecular Weight Multi-Drug Resistance Neisseria Nucleotides O Antigens Operon Organism Orphan Osmoregulation Pasteurella Pasteurella multocida Pathway interactions Phospholipids Plasmids Polymers Polysaccharides Positioning Attribute Production Proteins Reporting Research Structure Surface System Testing Thick Tissues Virulence Factors X-Ray Crystallography antimicrobial assault base building materials capsule cell envelope cell motility design detection method drug development genetic manipulation in vivo Model insight live cell imaging microbial nanodisk novel pathogen pathogenic bacteria pathogenic microbe periplasm prevent reconstitution restriction enzyme stoichiometry success thermophilic organism three dimensional structure tool

项目摘要

Essentially all living systems produce cell surface structures to rigidify cells, form protective coats, or facilitate cell adhesion and migration. Microbial ‘cell walls’ usually perform protective functions for survival under detrimental conditions, to reduce the efficacy of their host’s innate immune response, or to form 3-dimensional meshworks, called biofilms. Common building materials for these extracellular structures are polysaccharides that either function on their own or are integrated with other polymers into elaborate composite materials. Capsular polysaccharides (CPS) are abundant among Gram-negative and –positive bacteria. The polymers form dense extracellular structures that limit diffusion, aid in osmoregulation, and form thick protective coats around the cell. Some CPS mimic host glycans, thereby disguising potent pathogens under an immunologically invisible coat. The polymers are synthesized and deposited on the cell surface by two fundamentally different pathways. One assembles the polymer in the periplasm from short lipid-linked precursors and translocates it across the outer membrane (OM) concomitantly. In the ABC transporter- dependent pathway, however, the CPS is synthesized intracellularly on a lipid anchor and transported after its completion through a secretion system that spans the inner and the OM. The molecular and mechanistic mechanisms of both pathways remain poorly understood. To aid the development of novel antibiotic strategies, we seek to establish a detail structure-function analysis of the abundant ABC transporter-dependent CPS biosynthesis pathway. Our approach is two-pronged. First, we seek to establish a robust genetically tractable model system for CPS secretion (Aim 1A and B). Second, we will complement our functional analyses with detailed structural insights into the CPS ABC transporter (Aim 2), thereby providing the molecular basis for substrate recognition, CPS translocation, as well as interaction with periplasmic and OM transporter components. To this end, we engineered a standard E. coli laboratory strain to produce a polysaccharide capsule from plasmid-encoded components. The expressed operon contains 9 genes and each can be removed from its expression plasmid by standard restriction enzyme digestion. Further, we also developed a molecular probe enabling the detection of the synthesized capsule on the cell surface, thereby correlating CPS production with the expression of the biosynthetic machinery. To integrate our functional analyses with a 3D structure of the CPS ABC transporter, we purified a stable transporter in complex with its periplasmic subunit that likely stabilizes interactions with the OM pore. We will use cryo electron microscopy to determine the transporter’s structure in different nucleotide-bound states. Combined, our proposed research will provide the molecular basis for CPS secretion and lay the foundation for structure-guided drug development.

基本上所有的生命系统都会产生细胞表面结构来硬化细胞、形成保护层或促进微生物“细胞壁”通常在环境下发挥生存保护功能。有害条件，降低宿主先天免疫反应的功效，或形成 3 维这些细胞外结构的常见建筑材料是多糖。它们要么单独发挥作用，要么与其他聚合物集成成复杂的复合材料。荚膜多糖（CPS）在革兰氏阴性和阳性细菌中含量丰富。聚合物形成致密的细胞外结构，限制扩散，帮助渗透调节，并形成粘稠的细胞外结构一些 CPS 模仿宿主聚糖，从而将强效病原体隐藏在细胞周围。免疫学上看不见的涂层是由两种物质合成并沉积在细胞表面上的。一种是通过短脂质连接在周质中组装聚合物。前体并同时将其转运穿过外膜 (OM) 在 ABC 转运蛋白中。然而，CPS 是依赖于途径的，在细胞内合成在脂质锚上，并在其转运后进行运输。通过跨越内部和 OM 的分泌系统完成。这两种途径的机制仍知之甚少，以帮助开发新型抗生素策略。我们寻求对丰富的 ABC 转运蛋白依赖性 CPS 进行详细的结构功能分析生物合成途径。我们的方法是双管齐下的，首先，我们寻求建立一个强大的遗传易处理的模型系统。其次，我们将通过详细的结构来补充我们的功能分析。深入了解 CPS ABC 转运蛋白（目标 2），从而为底物识别提供分子基础， CPS 易位，以及与周质和 OM 转运蛋白成分的相互作用。为此，我们设计了标准大肠杆菌实验室菌株来生产多糖胶囊表达的操纵子包含 9 个基因，每个基因都可以从质粒编码的组件中删除。通过标准限制酶消化其表达质粒此外，我们还开发了分子探针。能够检测细胞表面的合成胶囊，从而将 CPS 的产生与生物合成机制的表达。为了将我们的功能分析与 CPS ABC 转运蛋白的 3D 结构相结合，我们纯化了与其周质亚基复合的稳定转运蛋白可能稳定与 OM 孔的相互作用。我们将使用冷冻电子显微镜来确定不同核苷酸结合的转运蛋白的结构综合起来，我们提出的研究将为 CPS 分泌提供分子基础并奠定基础。结构引导药物开发的基础。