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产生细胞表面结构，以使细胞硬化，形成保护性涂层，或促进细胞生长。 细胞粘附和迁移。微生物的“细胞壁”通常在环境中起保护作用， 有害的条件下，以降低其宿主的先天免疫反应的功效，或形成三维 称为生物膜的网状物。这些细胞外结构的常见建筑材料是多糖 它们要么自己发挥作用，要么与其他聚合物结合成复杂的复合材料。 荚膜多糖（CPS）在革兰氏阴性菌和革兰氏阳性菌中含量丰富。的 聚合物形成致密的细胞外结构，限制扩散，有助于细胞调节， 细胞周围的保护层。一些CPS模仿宿主聚糖，从而在一个特定的环境下伪装有效的病原体。 免疫隐形衣。该聚合物是通过两种方法合成并沉积在细胞表面的。 根本不同的路径。一种是在周质中组装聚合物， 前体和易位它跨越外膜（OM）伴随。在ABC运输机中- 然而，CPS是在细胞内在脂质锚上合成的，并在其释放后转运。 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转运蛋白依赖性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，我们寻求建立一个强大的遗传易处理的模型系统 CPS分泌（目标1A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易位，以及与周质和OM转运蛋白组分的相互作用。 为此，我们设计了一个标准的E。大肠杆菌实验室菌株生产多糖胶囊 从质粒编码的成分。表达的操纵子含有9个基因，每个基因都可以从 通过标准限制性内切酶消化其表达质粒。此外，我们还开发了一种分子探针， 使得能够检测细胞表面上的合成胶囊，从而将CPS生产与 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，我们纯化了一个 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 states.结合这些，我们的研究将为CPS的分泌提供分子基础，并为将来的研究奠定基础。 结构导向药物开发基金会。