Synthesis, secretion and assembly of extracellular complex carbohydrates in Gram-negative bacteria

革兰氏阴性菌胞外复合碳水化合物的合成、分泌和组装

• 批准号: 10330628
• 负责人: Jochen Zimmer
• 金额: $ 42.19万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-01-01 至 2026-12-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10330628
• 关键词: ATP-Binding Cassette Transporters; Address; Anabolism; Architecture; Bacteria; Biochemical; Biochemistry; Bioinformatics; Biological; Biological Models; Biophysics; Biopolymers; Carbohydrates; Cell Communication; Cell surface; Cells; Cellulose; Complex; Cryoelectron Microscopy; Deposition; Enterobacteriaceae; Enzymes; Escherichia coli; Extracellular Matrix; Glucans; Glycobiology; Gram-Negative Bacteria; Health; Human; Hydrophobicity; In Vitro; Life; Lipids; Lipopolysaccharides; Mediating; Membrane; Microbial Biofilms; Molecular; Molecular Biology; Molecular Weight; Nosocomial Infections; O Antigens; Organism; Osmoregulation; Pathologic Processes; Pathway interactions; Physiological Processes; Plants; Play; Polymers; Polysaccharides; Process; Research; Roentgen Rays; Role; Structure; System; Teichoic Acids; Viral; Virulence Factors; Water; biomaterial development; cellulose synthase; drug development; extracellular; human pathogen; insight; microbial; novel therapeutics; pathogen; periplasm; phosphoethanolamine; polypeptide; reconstitution; structural biology

Complex carbohydrates are essential biopolymers ubiquitously expressed in all kingdoms of life. On cell surfaces, they usually perform architectural functions by fortifying the cell boundary, aiding in osmo-regulation, defining an extracellular matrix, and mediating cell-to-cell communications, among many other roles. We understand fairly well how polypeptides are transported across or integrated into biological membranes. Similar mechanisms for polysaccharides, which range from acidic to water-insoluble hydrophobic polymers, remain mostly unexplored, despite playing critical roles in many physiological and pathological processes. My research seeks to fill this gap. We integrate structural biology approaches with biochemistry, glycobiology, biophysics, bioinformatics, and molecular biology to delineate how high molecular weight complex carbohydrates are synthesized and deposited on the cell surface. Leveraging microbial, viral, plant and vertebrate model systems, we provide atomistic descriptions of mechanistically distinct secretion systems. Understanding these processes on a molecular level aids novel drug and biomaterial developments. This proposal combines two research directions on microbial extracellular polysaccharides synthesized and secreted by fundamentally different mechanisms. First, cellulose, a linear glucose polymer, is an important biofilm component of many enterobacteria, including E. coli. Biofilms pose a particular threat to human health, causing ~80% of nosocomial infections. Cellulose is synthesized by a ‘synthase-dependent’ pathway in which a membrane-embedded enzyme synthesizes and secretes the polymer. Most enterobacteria modify cellulose with phosphoethanolamine during secretion to stabilize it on the cell surface. We provided the first insights into the molecular organization of the supramolecular cellulose synthase complex. Our future research direction will address how cellulose biosynthesis is controlled, how cellulose is modified in the periplasm, and how it is transported across the periplasm and the outer membrane. Second, Gram-negative bacteria are protected by an outer membrane containing lipopolysaccharides (LPS) in the extracellular leaflet. LPS molecules contain variable O antigen polysaccharides that significantly extend the bacteria’s outermost protective coat and provide survival benefits to many human pathogens. Prior to attachment to the conserved LPS core, O antigens are completely assembled on a lipid linker inside the cell and transported to the periplasm by an ATP- fueled ABC transporter, called WzmWzt. We currently lack any mechanistic insights into how ABC transporters translocate biopolymers, such as polysaccharides, teichoic acids and polypeptides, which are all potent virulence factors. Our X-ray and cryo electron microscopy structures of WzmWzt in O antigen-free states provided the first insights into the transporter’s function. We now seek to determine the mechanism by which O antigens are secreted. This will be accomplished biochemically by reconstituting O antigen translocation in vitro, and structurally by determining snapshots of WzmWzt during substrate translocation.

复合碳水化合物是在所有生命王国中普遍表达的基本生物聚合物。打开单元格 表面，它们通常通过加固细胞边界，帮助渗透调节， 定义细胞外基质，以及调节细胞间的通信，以及许多其他角色。我们 很好地了解多肽是如何通过生物膜转运或整合到生物膜中的。类似 多糖类，从酸性到不溶于水的疏水聚合物的作用机制仍然存在。 尽管在许多生理和病理过程中发挥着关键作用，但大多数情况下仍未被探索。我的 研究试图填补这一空白。我们将结构生物学方法与生物化学、糖生物学、 生物物理学、生物信息学和分子生物学来描述高分子量的复杂程度 碳水化合物被合成并沉积在细胞表面。利用微生物、病毒、植物和 脊椎动物模型系统，我们提供了机械上不同的分泌系统的原子描述。 在分子水平上了解这些过程有助于新药和生物材料的开发。这 建议结合微生物胞外多糖合成和合成两个研究方向 由根本不同的机制分泌。首先，纤维素是一种线性葡萄糖聚合物，是一种重要的 许多肠道细菌的生物膜成分，包括大肠杆菌。生物膜对人类健康构成了特别的威胁， 造成约80%的医院感染。纤维素是通过“合成酶依赖”途径合成的，在该途径中 包埋在膜中的酶合成并分泌聚合物。大多数肠杆菌对纤维素进行修饰 在分泌过程中使用磷乙醇胺，使其稳定在细胞表面。我们提供了第一批关于 超分子纤维素合成酶复合体的分子组织。我们未来的研究方向是 介绍如何控制纤维素的生物合成，如何在周质中修饰纤维素，以及它是如何 通过周质和外膜运输。其次，革兰氏阴性细菌受到 胞外小叶中含有脂多糖的外膜。脂多糖分子中含有 可变O抗原多糖，显著延长细菌最外层的保护层，并 为许多人类病原体提供生存益处。在连接到保守的脂多糖核心O之前 抗原完全组装在细胞内的脂质连接物上，并通过ATP-1转运到周质- 加了燃料的ABC运输机，叫做WzmWzt。我们目前缺乏任何机械性的见解来了解ABC运输器是如何 转移生物聚合物，如多糖、磷壁酸和多肽，这些都是有效的 毒力因素。WzmWzt在O无抗原状态下的X射线和低温电子显微镜结构 提供了对传送器功能的第一次洞察。我们现在试图确定O的机制 抗原是分泌出来的。这将通过重组O抗原转位在生物化学上完成 体外，并通过确定WzmWzt在底物转运期间的快照在结构上。