ABC transporter-mediated secretion of capsular polysaccharides

ABC 转运蛋白介导的荚膜多糖分泌

• 批准号: 10287699
• 负责人: Jochen Zimmer
• 金额: $ 22.52万
• 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 2023-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10287699
• 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.

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 细胞粘附和迁移。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 网状结构，称为生物膜。这些细胞外结构的常见构建材料是多糖 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。这 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 细胞周围有保护层。一些 CPS 模仿宿主聚糖，从而掩盖潜在的病原体 免疫学隐形外套。聚合物由两个合成并沉积在细胞表面 根本上不同的途径。一种在周质中由短脂质连接组装聚合物 前体并同时将其易位穿过外膜（OM）。在 ABC 运输机中- 然而，CPS 是依赖途径，在脂质锚上在细胞内合成，并在其后转运。 通过跨越内部和 OM 的分泌系统完成。分子和机制 这两种途径的机制仍知之甚少。为了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模型系统 用于 CPS 分泌（目标 1A 和 B）。其次，我们将用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 来自质粒编码的成分。表达的操纵子包含 9 个基因，每个基因都可以从 其表达质粒经标准限制性酶消化。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 州。结合起来，我们提出的研究将为 CPS 分泌提供分子基础，并奠定 结构引导药物开发的基础。