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通过PI发现的9种组成酶的保守途径在内膜的细胞质表面合成。在核心寡糖附着后，核心-脂质A复合物通过ABC转运蛋白MsbA翻转到内膜的外表面，其中O-抗原聚合物附着。新生LPS随后运输到外膜涉及第二个ABC转运蛋白和两个外膜蛋白，由lpt基因编码。脂质A的许多额外的共价修饰可以在其从内膜的外表面转运到外膜的过程中发生。因此，脂质A修饰酶是细菌包膜内LPS运输的优秀报告基因。然而，修饰系统在不同的革兰氏阴性菌之间是相当可变的，并且通常受环境因素的调节。虽然不需要生长，脂质A修饰酶可以调节一些病原体的毒力。编码脂质A修饰酶的基因的突变或异源表达促进了脂质A结构在不同细菌菌株中的再工程化，并且可以实现新疫苗的开发，如PI最近对新杀弗朗西斯菌（一种小鼠特异性人兔热病模型生物体）的遗传研究所示。鉴于Francisella的重要性和它含有许多前所未有的脂质A修饰酶的事实，下一个资助期的具体目标是：1）F. novicida; 2）F. 3）阐明了F. novicida;和4）表征修饰酶，其羟基化或氧化Kdo 2-脂质A，一种确定的LPS亚结构，支持E.大肠杆菌生长并有效激活Toll样受体4（TLR 4）。不同修饰酶及其结构基因的可用性将使新型Kdo 2-lipid A衍生物在E.杆菌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本见解。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”;它们在光学显微镜下表现为淡粉色的杆状物，因为它们无法吸收紫色染料，称为革兰氏染色。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，都含有一层外膜，使它们不透某些染料和抗生素。革兰氏阴性菌外膜的外表面含有大量称为脂多糖（LPS）的独特物质，其由脂质A固定。组装LPS的脂质A锚的酶是保守的，并且是用于设计具有针对革兰氏阴性病原体的活性的新抗生素的极好靶标，所述革兰氏阴性病原体已经对商业抗生素产生抗性。此外，通过操纵编码组装LPS的脂质A结构域的酶的基因来修饰活细菌中的脂质A结构可用于减毒细菌病原体，使得它们可用作疫苗。

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.