Biosynthesis And Function Of Lipopolysaccharides

脂多糖的生物合成和功能

• 批准号: 7478659
• 负责人: Christian R Raetz
• 金额: $ 59.27万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-09-01 至 2010-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7478659
• 关键词: ATP-Binding Cassette Transporters; Acylation; Agonist; Anabolism; Antibiotics; Bacteria; Base Composition; Binding; Biochemical Genetics; Biological Assay; Blood Vessels; Cells; Cessation of life; Ciprofloxacin; Collaborations; Condition; Cytoplasm; Development; Disaccharides; Endothelial Cells; Endotoxins; Engineering; Enzymatic Biochemistry; Enzyme Gene; Enzymes; Escherichia coli; Event; Facility Construction Funding Category; Francisella; Genes; Glucosamine; Glycolipids; Gram-Negative Bacteria; Grant; Growth; Immune; In Vitro; Infection; Inflammatory; Interleukin-1 Receptors; Interleukin-6; Laboratories; Lipid A; Lipopolysaccharide Biosynthesis Pathway; Lipopolysaccharides; Localized; Macrophage Activation; Mass Spectrum Analysis; Measures; Mediator of activation protein; Membrane; Membrane Proteins; Modification; Monitor; Multiple Organ Failure; Mutate; O Antigens; Oligosaccharides; Pathway interactions; Phospholipids; Phosphoric Monoester Hydrolases; Polymyxin Resistance; Positioning Attribute; Production; Protein Family; Proteins; Reaction; Receptor Signaling; Reporter; Resistance; Resolution; Salmonella; Salmonella typhimurium; Scheme; Septic Shock; Structure; Surface; System; TLR4 gene; Therapeutic; Tumor Necrosis Factor-alpha; Vesicle; Virulence; Work; cell envelope; design; expression cloning; genetic analysis; human TNF protein; in vitro Assay; inhibitor/antagonist; inorganic phosphate; insight; interest; member; membrane assembly; membrane biogenesis; mutant; novel; novel vaccines; pathogen; periplasm; protein transport; response; sugar; trafficking; vaccine development

DESCRIPTION (provided by applicant): The outer surface of Gram-negative bacteria is covered with a remarkable, macromolecular glycolipid known as lipopolysaccharide (LPS), the hydrophobic anchor of which is lipid A. In Escherichia coli, lipid A consists of a hexa-acylated disaccharide of glucosamine with phosphate groups at the 1 and 4' positions. The minimal LPS substructure required for growth is lipid A with one or two additional sugars. Inhibition of any of the first seven enzymes that synthesize lipid A is lethal. These enzymes are expressed constitutively and are conserved in virtually all Gram-negative bacteria. Potent inhibitors have recently been discovered that target the second enzyme (LpxC) of pathway, with antibiotic activity comparable to ciprofloxacin. Lipid A (also known as endotoxin) is the active component of LPS that stimulates immune cells. During severe Gram-negative infections, the lipid A moiety of LPS, shed from bacteria, can cause excessive activation of macrophages and endothelial cells. Over-production of inflammatory mediators, such as TNF- alpha, IL-1beta, IL-6 and other proteins, damages small blood vessels. A full response to endotoxin leads to Gram- negative septic shock with multiple organ failure and death. A therapeutic approach has emerged with the discovery that certain lipid A-like molecules, including some precursors, are endotoxin antagonists. The signaling receptor for lipid A is the TLR4 protein, which is distantly related to the IL-1 receptor. TLR4 and its subunit MD2 on immune cells can discriminate between agonists and antagonists. One can re-engineer the lipid A of viable bacteria to be a TLR4 antagonist or a partial agonist, with potential applications in new vaccine development. In previous work, the P. I. identified the 9 constitutive and 10 inducible enzymes in E. coli and Salmonella, needed for lipid A biosynthesis and modification, respectively. The genes encoding these enzymes were also identified, primarily by expression cloning. The essential ABC transporter MsbA, which is closely related to the eukaryotic Mdr proteins, flips newly made LPS across the inner membrane, and is also required for LPS and phospholipid export to the outer membrane. The specific aims for the coming grant period are: I) the biochemical and genetic analysis of potent new LpxA and LpxC inhibitors; II) purification and characterization of the membrane enzymes LpxB, LpxK, KdtA, LpxL, and ArnT of lipid A biosynthesis; III) development of quantitative assays for the assembly of E. coli LPS core sugars and O-antigen; and IV) the design of in vitro systems for measuring LPS flip-flop and transport. These studies should accelerate the development of antibiotics that target the lipid A system and provide fundamental insights into the mechanisms of bacterial membrane assembly.

描述（由申请人提供）：革兰氏阴性细菌的外表面被称为脂多糖（LPS）的显着的，大分子糖脂覆盖，其疏水性锚固是脂质A。在Escherichia Coli中，脂质的脂质A组成了Hexa-acylated Disacame and glucaChathe and glucataCheAmine and glucataCheAmine flucatation and flucate flucatation and flucatation and flucatation and chucchate flucataCheAmine and chuctAcation。生长所需的最小LPS子结构是脂质A，另外一两个糖。抑制合成脂质A的前七种酶中的任何一种是致命的。这些酶是组成型表达的，并且在几乎所有革兰氏阴性细菌中都保存。最近发现有效的抑制剂靶向途径的第二酶（LPXC），其抗生素活性与环丙沙星相当。脂质A（也称为内毒素）是刺激免疫细胞的LP的活性成分。在严重的革兰氏阴性感染中，从细菌中脱离的LPID脂质部分会导致巨噬细胞和内皮细胞的过度激活。炎症介质（例如TNF-Alpha，IL-1Beta，IL-6和其他蛋白质）的过量生产会损害小血管。对内毒素的全面反应会导致多器官衰竭和死亡导致革兰氏阴性休克。发现某些脂质A样分子（包括某些前体）是内毒素拮抗剂的发现，已经出现了一种治疗方法。脂质A的信号传导受体是TLR4蛋白，它与IL-1受体遥远相关。 TLR4及其在免疫细胞上的亚基MD2可以区分激动剂和拮抗剂。可以将可行细菌的脂质A重新设计为TLR4拮抗剂或部分激动剂，并在新的疫苗开发中使用潜在的应用。在先前的工作中，P。I.分别确定了大肠杆菌和沙门氏菌中的9种本构和10种诱导酶，分别用于脂质A生物合成和修饰所需的。编码这些酶的基因也主要通过表达克隆鉴定。与真核MDR蛋白密切相关的必需ABC转运蛋白MSBA将新制成的LPS跨越内膜，也需要LPS和磷脂导出到外膜。即将到来的赠款期的具体目的是：i）有效的新LPXA和LPXC抑制剂的生化和遗传分析； ii）脂质酶的纯化和表征LPXB，LPXK，KDTA，LPXL和ARNT脂肪A脂肪生物合成； iii）开发大肠杆菌核心糖和O-抗原的定量测定； iv）用于测量LPS触发器和传输的体外系统的设计。这些研究应加快针对脂质A系统的抗生素的发展，并提供对细菌膜组装机制的基本见解。