Lipid A Modification Systems in Gram-Negative Bacteria

说明(申请人提供)：脂多糖(LPS)的脂类A部分形成了大多数革兰氏阴性细菌外膜的外单分子层。由等电点发现的九种组成酶的保守途径在细胞内膜表面合成了大肠杆菌脂质A。在核心低聚糖附着后，核心-脂类A复合体被ABC转运体MSBA翻转到内膜的外表面，O-抗原聚合物在那里附着。新生的内毒素随后运输到外膜涉及第二个ABC转运体和两个外膜蛋白，由LPT基因编码。在脂类A从内膜外表面到外膜的运输过程中，可能会发生许多额外的共价修饰。因此，类脂A修饰酶是细菌被膜内脂多糖运输的极佳报告。然而，在不同的革兰氏阴性菌之间，修饰系统是非常不同的，并且经常受到环境因素的调节。虽然不是生长所必需的，但脂质A修饰酶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促进了不同细菌株中脂质A结构的重新设计，并可以使新疫苗的开发成为可能，正如PI最近对新弗朗西斯菌(Francisella Novicida)的遗传学研究所证明的那样，新弗朗西氏菌是一种小鼠特有的人类图拉热症模式生物。鉴于方济氏菌的重要性，以及它含有许多前所未有的类脂A修饰酶的事实，下一个资助期的具体目标将是：1)新生链球菌脂A磷酸酶和糖基转移酶的纯化和鉴定；2)新链球菌脂多糖核心组装酶的生化和遗传分析；3)阐明新链球菌游离类脂A与脂多糖含量的调节；以及4)羟化或氧化Kdo2-类脂A的修饰酶的特征，脂多糖是一种支持大肠杆菌生长并潜在激活Toll样受体4(TLR4)的亚结构。不同的修饰酶及其结构基因的可获得性将使在大肠杆菌中大规模、组合地合成新型的KDO2-脂类A衍生物成为可能。除了为疫苗开发创造新的机会外，这些研究还将提供对A类脂生物、化学和外膜生物发生的基本见解。与公共卫生相关：人类所有的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看起来像淡粉色的棒状，因为它们不能吸收一种称为革兰氏染色的紫色染料。这类细菌包括所有的大肠杆菌、沙门氏菌、假单胞菌和方济各氏菌，它们含有一层外膜，使它们对某些染料和抗生素不能渗透。革兰氏阴性菌外膜的外表面含有大量被脂类A固定的独特物质，称为脂多糖(LPS)。组成脂类A锚的酶是保守的，是设计具有抗药性的革兰氏阴性病原体的新抗生素的极佳靶标。此外，通过操纵编码组成脂蛋白A结构域的酶的基因来修饰活细菌中的脂蛋白A结构，有助于减弱细菌病原体，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.