Biosynthesis And Function Of Lipopolysaccharides

描述(申请人提供)：革兰氏阴性菌的外表面覆盖着一种显著的大分子糖脂，称为脂多糖(LPS)，其疏水锚是脂类A。在大肠杆菌中，脂类A由氨基葡萄糖的六酰化二糖组成，在1和4‘位有磷酸基团。生长所需的最小内毒素亚基是含有一到两种额外糖的脂类A。抑制合成类脂A的前七种酶中的任何一种都是致命的。这些酶是结构性表达的，在几乎所有的革兰氏阴性细菌中都是保守的。最近发现了针对途径第二酶(LpxC)的有效抑制剂，其抗菌活性与环丙沙星相当。脂蛋白A(又称内毒素)是脂多糖中刺激免疫细胞的活性成分。在严重的革兰氏阴性感染期间，细菌释放出的脂蛋白A部分会导致巨噬细胞和内皮细胞的过度激活。过度产生炎症介质，如TNF-α、IL-1β、IL-6和其他蛋白质，会损害小血管。对内毒素的完全反应会导致革兰氏阴性败血症休克，并伴有多器官衰竭和死亡。一种治疗方法已经出现，发现某些类脂A分子，包括一些前体，是内毒素拮抗剂。脂质A的信号受体是TLR4蛋白，它与IL-1受体关系密切。免疫细胞上的TLR4及其亚基MD2可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TLR4拮抗剂或部分激动剂，在新疫苗开发中具有潜在的应用。在之前的工作中，P.I.鉴定了大肠杆菌和沙门氏菌中分别需要的9种组成酶和10种诱导酶，这两种酶分别是脂质A生物合成和修饰所需的。编码这些酶的基因也被鉴定出来，主要是通过表达克隆。与真核细胞MDR蛋白密切相关的基本ABC转运蛋白MSBA将新合成的内毒素翻转到内膜，也是内毒素和磷脂输出到外膜所必需的。未来资助期的具体目标是：i)有效的新LpxA和LpxC抑制剂的生化和遗传分析；ii)脂类A生物合成的膜酶LpxB、LpxK、KdtA、LpxL和Arnt的纯化和性质；iii)建立大肠杆菌内毒素核心糖和O-抗原组装的定量检测方法；以及IV)体外检测脂多糖的触发和转运系统。这些研究将加速针对脂质A系统的抗生素的开发，并为细菌膜组装的机制提供基本的见解。

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.