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: 10543793
• 负责人: Jochen Zimmer
• 金额: $ 54.07万
• 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/10543793
• 关键词: ATP-Binding Cassette Transporters; 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; fortification; 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.

复杂碳水化合物是生命各个领域中普遍存在的重要生物聚合物。在电池上 它们通常通过强化细胞边界、帮助渗透调节来发挥建筑功能， 定义细胞外基质、介导细胞间通讯等许多作用。我们 很好地理解多肽如何跨过生物膜转运或整合到生物膜中。相似的 多糖（从酸性到水不溶性疏水性聚合物）的机制仍然存在 尽管在许多生理和病理过程中发挥着关键作用，但大部分尚未被探索。我的 研究旨在填补这一空白。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复合物 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物和生物材料的开发。这 该提案结合了微生物胞外多糖合成和合成的两个研究方向 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 许多肠细菌（包括大肠杆菌）的生物膜成分。生物膜对人类健康构成特别威胁， 引起~80%的医院感染。纤维素是通过“合酶依赖性”途径合成的，其中 膜嵌入的酶合成并分泌聚合物。大多数肠杆菌修饰纤维素 在分泌过程中与磷酸乙醇胺结合以将其稳定在细胞表面。我们提供了第一手见解 超分子纤维素合酶复合物的分子组织。我们未来的研究方向将 解决纤维素生物合成如何控制、纤维素在周质中如何修饰以及它是如何进行的 穿过周质和外膜运输。其次，革兰氏阴性菌受到保护 细胞外小叶中含有脂多糖（LPS）的外膜。 LPS分子中含有 可变 O 抗原多糖，可显着延长细菌最外层的保护层， 为许多人类病原体提供生存益处。在连接到保守的 LPS 核心之前，O 抗原完全组装在细胞内的脂质接头上，并通过 ATP-转运至周质 为 ABC 运输机提供燃料，称为 WzmWzt。我们目前缺乏任何关于 ABC 运输者如何机制的见解 转移生物聚合物，例如多糖、磷壁酸和多肽，这些都是有效的 毒力因素。我们的 X 射线和冷冻电子显微镜结构的 WzmWzt 无 O 抗原状态 首次深入了解转运蛋白的功能。我们现在寻求确定 O 的机制 抗原被分泌。这将通过在生物化学中重建 O 抗原易位来完成 体外和结构上通过确定底物易位过程中 WzmWzt 的快照。