Biosynthesis And Function Of Lipopolysaccharides

描述（由申请人提供）：革兰氏阴性菌的外表面覆盖有一种显著的大分子糖脂，称为脂多糖（LPS），其疏水性锚是脂质A。在大肠杆菌中，脂质A由葡糖胺的六酰化二糖组成，在1和4'位置具有磷酸基团。生长所需的最小LPS亚结构是具有一个或两个额外糖的脂质A。抑制前七种合成脂质A的酶中的任何一种都是致命的。这些酶组成型表达，并在几乎所有革兰氏阴性菌中保守。最近发现了靶向途径的第二种酶（LpxC）的有效抑制剂，其抗生素活性与环丙沙星相当。脂质A（也称为内毒素）是LPS的活性成分，可刺激免疫细胞。在严重的革兰氏阴性感染期间，从细菌脱落的LPS的脂质A部分可引起巨噬细胞和内皮细胞的过度活化。炎症介质如TNF-α、IL-1 β、IL-6和其他蛋白质的过度产生会损害小血管。对内毒素的完全反应会导致革兰氏阴性菌感染性休克，并伴有多器官衰竭和死亡。随着某些脂质A样分子（包括一些前体）是内毒素拮抗剂的发现，已经出现了治疗方法。脂质A的信号传导受体是TLR 4蛋白，其与IL-1受体关系较远。免疫细胞上的TLR 4及其亚基MD 2可以区分激动剂和拮抗剂。人们可以将活细菌的脂质A重新设计为TLR 4拮抗剂或部分激动剂，在新疫苗开发中具有潜在的应用。在以前的工作中，私人侦探。鉴定了E.大肠杆菌和沙门氏菌，分别需要脂质A的生物合成和修饰。还鉴定了编码这些酶的基因，主要是通过表达克隆。与真核生物Mdr蛋白密切相关的必需ABC转运蛋白MsbA将新产生的LPS翻转穿过内膜，并且也是LPS和磷脂输出到外膜所需的。本研究的主要目的是：（1）对LpxA和LpxC抑制剂进行生物化学和遗传学分析;（2）对脂质A生物合成中的膜酶LpxB、LpxK、KdtA、LpxL和ArnT进行纯化和鉴定;（3）对E. coli LPS核心糖和O-抗原;和IV）用于测量LPS触发和转运的体外系统的设计。这些研究将加速靶向脂质A系统的抗生素的开发，并为细菌膜组装机制提供基本见解。

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.