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.

基本上，所有的生命系统都会产生细胞表面结构，以使细胞变得坚硬，形成保护层，或促进 细胞黏附和迁移。微生物的“细胞壁”通常起到保护生命的作用 有害的条件，以降低其宿主的先天免疫反应的效力，或形成三维 网状结构，称为生物膜。这些胞外结构的常见建筑材料是多糖 这些聚合物要么单独发挥作用，要么与其他聚合物集成在一起，形成精致的复合材料。 革兰氏阴性菌和革兰氏阳性菌中都含有丰富的荚膜多糖。这个 聚合物形成致密的胞外结构，限制扩散，帮助渗透调节，并形成稠密的 牢房周围穿着防护服。一些CP模仿宿主多糖，从而将潜在的病原体伪装在 免疫隐形外套。聚合物是通过两种方式合成并沉积在细胞表面的。 根本不同的路径。一种是从短脂连接的聚合物在周质中组装 前体，并同时将其转移到外膜(OM)。在ABC运输机里- 然而，依赖的途径，CPS是在脂质锚上细胞内合成的，并在其 通过一个跨越内部和OM的分泌系统来完成。分子和机械论 这两条途径的机制仍然知之甚少。为了帮助开发新的抗生素策略， 我们试图建立丰富的ABC转运蛋白依赖的CPS的详细结构-功能分析 生物合成途径。 我们的方法是双管齐下的。首先，我们寻求建立一个健壮的、遗传上易于处理的模型系统 用于CPS分泌(目标A和B)。其次，我们将用详细的结构分析来补充我们的功能分析 深入了解CPS ABC转运蛋白(AIM 2)，从而为底物识别提供分子基础， CPS转运，以及与周质和OM转运蛋白成分的相互作用。 为此，我们设计了一种标准的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 其表达载体经标准限制性内切酶酶切。此外，我们还开发了一种分子探针 使得能够在细胞表面检测合成的胶囊，从而将CPS的产生与 生物合成机械的表现。 为了将我们的功能分析与CPS ABC转运蛋白的3D结构相结合，我们纯化了一个 稳定的转运蛋白与其周质亚基形成复合体，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 各州。综上所述，我们的研究将为CPS分泌提供分子基础，并为CPS分泌奠定基础 以结构为导向的药物开发基金会。