Lipid A Modification Systems in Gram-Negative Bacteria

革兰氏阴性细菌中的脂质 A 修饰系统

• 批准号: 7900959
• 负责人: Christian R Raetz
• 金额: $ 38.61万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-07-20 至 2013-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7900959
• 关键词: ATP-Binding Cassette Transporters; Anabolism; Animal Model; Antibiotics; Antimicrobial Cationic Peptides; Attenuated; Bacteria; Biochemical Genetics; Biology; Cells; Chemistry; Complex; Core Assembly; Cytoplasm; Development; Divalent Cations; Dyes; Engineering; Environmental Risk Factor; Enzyme Gene; Enzymes; Escherichia coli; Francisella; Francisella tularensis; Galactosamine; Gene Expression; Genes; Genetic; Glucose; Gram' s stain; Gram-Negative Bacteria; Grant; Growth; Human; Hydrolase; Laboratories; Life; Light Microscope; Link; Lipid A; Lipopolysaccharide Biosynthesis Pathway; Lipopolysaccharides; Mannose; Membrane; Membrane Lipids; Membrane Proteins; Mixed Function Oxygenases; Modification; Monitor; Mus; Mutate; Mutation; O Antigens; Oligosaccharides; Organism; Oxidases; Pathway interactions; Phosphoric Monoester Hydrolases; Polymers; Pseudomonas; Regulation; Reporter; Resistance; Rhizobium; Role; Salmonella; Salmonella typhimurium; Structural Genes; Structure; Surface; System; Transferase; Tularemia; Vaccine Research; Vaccines; Virulence; Work; Yersinia pestis; aminoarabinose; base; combinatorial; design; genetic analysis; glycosyltransferase; inorganic phosphate; insight; macrophage; membrane biogenesis; monolayer; mutant; novel; novel vaccines; pathogen; periplasm; phosphoethanolamine; public health relevance; retinal rods; routine Bacterial stain; sugar; toll-like receptor 4; trafficking; vaccine development

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.

描述（由申请人提供）：脂质的脂多糖（LPS）部分形成了大多数革兰氏阴性细菌的外膜的外部单层。大肠杆菌脂质A通过9种本构酶的保守途径在内膜的细胞质表面合成，这是由PI发现的。核寡糖附着后，核脂质A复合物通过ABC转运蛋白MSBA翻转到内膜的外表面，在该膜上附着O-抗原聚合物。随后将新生LPS运输到外膜的运输涉及第二个ABC转运蛋白和两个外膜蛋白，该蛋白质由LPT基因编码。在其从内膜的外表面到外膜的转运期间，可能发生许多其他的共价修饰。因此，脂质修饰酶是细菌包膜内LPS运输的出色记者。但是，修饰系统在不同的革兰氏阴性生物之间是很大的变化，并且通常受环境因素的调节。尽管不需要生长，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多种细菌菌株中脂质A结构的重新设计，并可以使PI的最新遗传研究与Francisella Novicida（小鼠 - 小鼠），小鼠Novicida，小鼠Novicida，小鼠Novicida（PI）进行了新的疫苗开发特定的模型生物体对人类t虫的生物体。鉴于Francisella的重要性及其包含许多前所未有的脂质修饰酶，因此即将到来的赠款期的具体目的是：1）脂质的磷酸酶A磷酸酶和NOVICIDA的糖基转移酶的纯化和表征； 2）F。Novicida的LPS核心组装酶的生化和遗传分析； 3）阐明自由脂质A与Novicida的LPS含量的调节； 4）羟基盐酸或氧化KDO2-脂质A的修饰酶的表征，该脂质A（一种支持大肠杆菌生长的LPS下结构）的表征，并有效激活类似Toll的受体4（TLR4）。各种修饰酶及其结构基因的可用性将使大肠杆菌中的新型，KDO2-脂质A衍生物的大规模组合生物合成。除了为疫苗开发创造新的机会外，这些研究还将为脂质A的生物学，化学和外膜外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的一半被归类为“革兰氏阴性”；它们在光学显微镜中看起来像浅色的粉红色棒，因为它们无法占据紫色的染料，称为革兰氏污渍。这种细菌包括所有大肠杆菌，沙门氏菌，假单胞菌和弗朗西斯氏菌的菌株，其中包含一种外膜，使它们不受某些染料和抗生素的不可渗透。革兰氏阴性细菌的外膜的外膜外表面含有大量的独特物质，称为脂多糖（LPS），该物质由脂质A固定在位A。针对革兰氏阴性病原体的活性设计新抗生素的靶标，这些抗生素对商业抗生素具有抗药性。此外，通过操纵编码组装脂质A LP的酶的基因来修饰活细菌中的脂质A结构，可用于减弱细菌病原体，以便将其用作疫苗。