Molecular Biology of Unique Wall Lipids of Mycobacteria

描述（由申请人提供）：结核病仍然是可预防死亡的主要原因。耐多药结核病的自然传播以及恐怖分子可能利用这类菌株，使得发现抗真菌治疗的新靶点成为一项非常迫切的需要。细胞壁脂质构成了一个主要的物理和化学防御屏障，帮助病原体逃避宿主的防御和抗真菌药物。因此，合成这种对感染至关重要的独特脂质可以成为新的抗真菌药物的合适靶点。多甲基支链脂类如二真菌酰硫代苯酚（DIM）已被证明是毒力因子。阐明这些毒力因子生物合成过程中的生化反应和酶的性质是开发新药所必需的。为此，我们建议：阐明脂肪酶基因的功能及其在发病机制中的可能作用，a)表达和表征lip基因产物的催化能力，确定特定lip基因的破坏是否会导致缺乏特定类别的酰基脂或缺乏特定组的酯化脂肪酸，并确定突变是否影响毒力；b)直接检测所表达的lip基因产物能否在体外释放所表达合酶的酰基链。2. 阐明这些基因的生物学功能及其在毒力中的可能作用，a)表达这些基因并表征其产物的催化能力，b)。破坏这三个tes基因，用14c标记的醋酸盐和14c标记的丙酸盐作为放射性示踪剂测定其对脂质代谢的影响，并在小鼠模型中测定突变对病原体毒力的影响。3. 阐明wes基因的功能，a)表达wes基因并测试这些基因产物是否参与由大型合酶产生的甲基支链酸到最终受体羟基的酯化反应，b)确定破坏wes基因对脂质代谢的影响并测试表现出新的生化表型的突变体的毒力。4. a)阐明新亚基在催化合成甲基丙二酰辅酶a的酶中的作用，甲基丙二酰辅酶a是多种甲基支链毒力因子的组成部分；b)确定accD4和accD5的破坏是否影响结核分枝杆菌中丙酰辅酶a的羧化和甲基支链脂质合成及其毒力。c)确定琥珀酸盐作为甲基丙二醇辅酶a来源的可能作用。本研究结果可能为筛选化学文库提供信息和工具，从而发现针对结核分枝杆菌新靶点的新型候选药物，从而有助于对抗耐多药结核病。

DESCRIPTION (provided by applicant): Tuberculosis remains the leading cause of preventable deaths. Natural spread of multidrug resistant tuberculosis and the potential use of such strains by terrorists make discovery of new targets for antimycobacterial therapy a very critical need. Cell wall lipids constitute a major physical and chemical defensive barrier that helps the pathogen evade the host defenses and antimycobacterial drugs. Therefore, synthesis of such unique lipids that are critical for infection can be suitable targets for new antimycobacterial drugs. Multiple methyl branched lipids such as dimycocerosyl phthiocerol (DIM) have been shown to be virulence factors. Elucidation of the biochemical reactions and the nature of the enzymes involved in the biosynthesis of such virulence factors is required for developing novel drags. To this end we propose to: 1. Elucidate the functions of lipase genes and their possible role in pathogenesis, a) Express and characterize the catalytic capabilities of lip gene products and determine whether disruption of specific lip genes will result in the absence of a specific class of acyl-lipids or absence of a specific group of esterified fatty acids, and determine whether the mutations affect virulence, b) Directly test whether the expressed lip gene products can release the acyl chains from the expressed synthases in vitro. 2. Elucidate the biological function of tes genes and their possible role in virulence, a) Express tes genes and characterize the catalytic capabilities of their products, b). Disrupt the three tes genes, determine the effects on lipid metabolism by using 14C-labeled acetate and 14C-labeled propionate as radiotracers and determine the effect of the mutations on virulence of the pathogen in the murine model. 3. Elucidate the function of wes genes, a) Express the wes genes and test whether these gene products are involved in the esterification of the methyl branched acids generated by the large synthases to the hydroxyl groups in the ultimate acceptors, b) Determine the consequences of disrupting the wes genes on lipid metabolism and test the virulence of mutants that show novel biochemical phenotypes. 4. a) Elucidate the role of the novel ( subunit in the enzyme that catalyzes the synthesis of methylmalonyl-CoA, the building block for the multiple methyl branched virulence factors, b) Determine whether disruption of accD4 and accD5 affect propionyl-CoA carboxylation, and methyl branched lipid synthesis in M. tuberculosis and its virulence. c) Determine the possible role of succinate as a source for methylmalonyl-CoA. The results of this study are likely to give information and tools for screening chemical libraries to discover new types of drug candidates directed at novel targets in M. tuberculosis and thus help in combating multidrug resistant tuberculosis.