ABC transporter-mediated secretion of capsular polysaccharides

ABC 转运蛋白介导的荚膜多糖分泌

• 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
• 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

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.

本质上，所有生物系统都会产生细胞表面结构以固化细胞，形成保护性外套或制备 细胞粘合剂和迁移。微生物“细胞壁”通常执行受保护的功能以生存 有害条件，以降低宿主先天免疫反应的效率或形成3维 网状工作，称为生物膜。这些细胞外结构的常见建筑材料是多糖 该要么自行起作用，要么与其他聚合物集成到精美的复合材料中。 在革兰氏阴性和阳性细菌中，囊囊多糖（CPS）丰富。这 聚合物形成密集的细胞外结构，限制扩散，有助于渗透调节并形成厚 在细胞周围的保护层。一些CPS模拟宿主聚糖，从而掩盖了潜在的病原体 免疫学上看不见的外套。聚合物合成并沉积在细胞表面上 基本不同的途径。一个人通过短脂质连接组装在周期中的聚合物 前体并同时将其翻译在外膜（OM）上。在ABC转运蛋白中 但是，依赖途径，CP在脂质锚上细胞内合成，并在其后运输 通过跨越内部和OM的分泌系统完成。分子和机械 这两种途径的机制仍然鲜为人知。为了开发新型抗生素策略， 我们试图建立对丰富的ABC转运蛋白依赖性CP的详细结构 - 功能分析 生物合成途径。 我们的方法是两管齐的。首先，我们试图建立一个坚固的一般易处理的模型系统 用于CPS分泌（AIM 1A和B）。其次，我们将通过详细的结构完成功能分析 对CPS ABC转运蛋白的见解（AIM 2），从而提供了底物识别的分子基础， CPS运输，以及与周质和OM转运蛋白成分的相互作用。 为此，我们设计了标准的大肠杆菌实验室应变以产生多糖胶囊 来自质粒编码的成分。表达的歌剧包含9个基因，每个基因都可以从 其表达质粒通过标准限制酶消化。此外，我们还开发了一个分子探针 可以在细胞表面检测合成胶囊，从而将CPS的产生与 生物合成机械的表达。 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A 稳定的转运蛋白及其与其周质亚基可能稳定与OM孔相互作用。 我们将使用冷冻电子显微镜确定在不同核苷酸结合的转运蛋白的结构 国家。合并，我们提出的研究将为CPS分泌提供分子基础 结构引导的药物开发基金会。