Lipid A Modification Systems in Gram-Negative Bacteria

描述（由申请人提供）：脂多糖（LPS）的脂质A部分形成大多数革兰氏阴性菌外膜的外单层。大肠杆菌脂质A在细胞膜细胞质表面通过9种组成酶的保守途径合成，这些酶是由PI发现的。随着核心寡糖的附着，核心脂质A复合物被ABC转运体MsbA翻转到内膜的外表面，在那里o抗原聚合物被附着。随后，新生LPS向外膜的运输涉及第二个ABC转运蛋白和两个由lpt基因编码的外膜蛋白。脂质A在从内膜外表面到外膜的转运过程中可能发生许多额外的共价修饰。因此，脂质A修饰酶是细菌包膜内脂多糖运输的优秀报告者。然而，不同革兰氏阴性菌之间的修饰系统差异很大，往往受环境因素的调节。脂质A修饰酶虽然不是生长所必需的，但可以调节某些病原体的毒力。编码脂质A修饰酶的基因的突变或异源表达有助于在不同菌株中重新设计脂质A结构，并能够开发新的疫苗，正如PI最近对新弗朗西斯菌（一种人类土拉菌的小鼠特异性模式生物）的遗传研究所说明的那样。鉴于Francisella的重要性以及它含有许多前所未有的脂质A修饰酶的事实，即将到来的资助期的具体目标将是：1)F. novicida的脂质A磷酸酶和糖基转移酶的纯化和表征；2)新乳霉LPS核心组装酶的生化和遗传分析；3)阐明了游离脂质A与脂多糖含量的关系；4)羟基化或氧化Kdo2-脂质A的修饰酶的特性，Kdo2-脂质A是一种支持大肠杆菌生长并有效激活toll样受体4 （TLR4）的确定的LPS亚结构。多种修饰酶及其结构基因的可用性将使新型kdo2 -脂质A衍生物在大肠杆菌中大规模组合生物合成成为可能。除了为疫苗开发创造新的机会外，这些研究还将为脂质A生物学、化学和外膜生物发生提供基础见解。公共卫生相关性：所有人类细菌病原体中有一半被归类为“革兰氏阴性”；在光学显微镜下，它们看起来像淡粉色的杆状体，因为它们不能吸收一种叫做革兰氏染色剂的紫色染料。这类细菌，包括大肠杆菌、沙门氏菌、假单胞菌和弗朗西斯菌的所有菌株，都有一层外膜，使它们不被某些染料和抗生素渗透。革兰氏阴性菌外膜的外表面含有大量被称为脂多糖（LPS）的独特物质，该物质由脂质a保持在适当的位置。组装脂质a锚定LPS的酶是保守的，并且是设计具有抗革兰氏阴性病原体活性的新抗生素的极好靶点，这些病原体已经对商业抗生素产生耐药性。此外，通过操纵编码LPS的脂质A结构域的酶的基因来修饰活细菌的脂质A结构，有助于减毒细菌病原体，使其可以用作疫苗。

DESCRIPTION (provided by applicant): The lipid A moiety of lipopolysaccharide (LPS) forms the outer monolayer of the outer membrane of most Gram-negative bacteria. Escherichia coli lipid A is synthesized on the cytoplasmic surface of the inner membrane by a conserved pathway of nine constitutive enzymes, which were discovered by the PI. Following attachment of the core oligosaccharide, the core-lipid A complex is flipped to the outer surface of the inner membrane by the ABC transporter MsbA, where the O-antigen polymer is attached. Subsequent trafficking of nascent LPS to the outer membrane involves a second ABC transporter and two outer membrane proteins, encoded by the lpt genes. Many additional covalent modifications of lipid A may occur during its transit from the outer surface of the inner membrane to the outer membrane. Lipid A modification enzymes are therefore excellent reporters for LPS trafficking within the bacterial envelope. However, modification systems are quite variable between different Gram-negative organisms and are often regulated by environmental factors. Although not required for growth, the lipid A modification enzymes can modulate the virulence of some pathogens. Mutation or heterologous expression of the genes encoding the lipid A modification enzymes facilitates the re-engineering of lipid A structure in diverse bacterial strains and can enable the development of new vaccines, as illustrated by the PI's recent genetic studies with Francisella novicida, a mouse-specific model organism for human tularemia. Given the importance of Francisella and the fact that it contains many unprecedented lipid A modification enzymes, the specific aims for the coming grant period will be: 1) the purification and characterization of the lipid A phosphatases and glycosyltransferases of F. novicida; 2) the biochemical and genetic analysis of LPS core assembly enzymes of F. novicida; 3) the elucidation of the regulation of the free lipid A versus LPS content of F. novicida; and 4) the characterization of modification enzymes that hydroxylate or oxidize Kdo2- lipid A, a defined LPS substructure that supports E. coli growth and potently activates toll-like receptor 4 (TLR4). The availability of diverse modification enzymes and their structural genes will enable the large-scale, combinatorial biosynthesis of novel, Kdo2-lipid A derivatives in E. coli. In addition to creating new opportunities for vaccine development, these studies will provide fundamental insights into lipid A biology, chemistry and outer membrane biogenesis. PUBLIC HEALTH RELEVANCE: Half of all human bacterial pathogens are classified as "Gram-negative"; they appear as pale, pink rods in the light microscope because of their inability to take up a purple dye, called Gram-stain. Bacteria of this kind, which include all strains of Escherichia coli, Salmonella, Pseudomonas, and Francisella, contain an outer membrane that makes them impermeable to certain dyes and antibiotics. The outer surfaces of the outer membranes of Gram-negative bacteria contain large amounts of a unique substance known as lipopolysaccharide (LPS), which is held in place by lipid A. The enzymes that assemble the lipid A anchor of LPS are conserved and are excellent targets for designing new antibiotics with activity against Gram-negative pathogens that have become resistant to commercial antibiotics. In addition, the modification of lipid A structure in live bacteria by manipulation of the genes encoding the enzymes that assemble the lipid A domain of LPS are useful for attenuating bacterial pathogens so that they can be used as vaccines.