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.

复合碳水化合物是必需的生物聚合物，在所有生命王国中普遍表达。对细胞 表面，它们通常通过强化细胞边界，帮助细胞调节， 定义细胞外基质和介导细胞间通讯等。我们 我们非常了解多肽是如何穿过或整合到生物膜中的。类似 从酸性到不溶于水的疏水性聚合物的多糖的机制仍然存在， 尽管在许多生理和病理过程中起着关键作用，但大多未被探索。我 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 在脊椎动物模型系统中，我们提供了机械上不同的分泌系统的原子描述。 在分子水平上理解这些过程有助于新药和生物材料的开发。这 该提案结合了微生物胞外多糖合成和 由不同的机制分泌首先，纤维素，一种线性葡萄糖聚合物，是一种重要的 许多肠细菌的生物膜成分，包括E.杆菌生物膜对人类健康构成特别的威胁， 导致约80%的医院感染。纤维素是通过一种“依赖于淀粉酶”的途径合成的， 膜包埋的酶合成并分泌聚合物。大多数肠杆菌会修饰纤维素 在分泌过程中用磷酸乙醇胺将其稳定在细胞表面。我们提供了关于 超分子纤维素合酶复合物的分子结构。我们未来的研究方向将 解决纤维素生物合成是如何控制的，纤维素是如何在周质中被修饰的，以及它是如何在细胞质中被修饰的。 通过周质和外膜运输。第二，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分子含有 可变的O抗原多糖，其显著延长细菌的最外层保护衣， 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转运到周质， 燃料ABC运输机，叫WzmWzt我们目前缺乏对ABC转运蛋白 转运生物聚合物，如多糖、磷壁酸和多肽，它们都是有效的 毒力因子我们的WzmWzt在无O抗原状态下的X射线和冷冻电子显微镜结构 首次揭示了这种转运蛋白的功能我们现在试图确定O 抗原被分泌。这将通过在细胞中重建O抗原易位来生化地完成。 体外和结构上通过确定快照的WzmWzt在底物易位。