MOLECULAR BIOLOGY OF UNIQUE WALL LIPIDS OF MYCOBACTERIA

描述(改编自申请人的摘要)：复兴 与艾滋病相关的结核病和其他分枝杆菌感染 在全球范围内威胁人类健康。耐药结核病的出现使它 对发现新的药物靶点至关重要。分枝杆菌细胞壁含有一种 多种脂类与多种甲基支链脂肪酸的关系 是致病分枝杆菌所特有的。这些脂质被认为是 在病原菌逃避防御的能力中发挥重要作用 主人的反应。这些独特的脂类的生物合成 多甲基分枝细胞壁脂可为新的 抗分枝杆菌药物。为了阐明这一过程中涉及的机制 这些脂质的生物合成并测试它们在体内的可能功能 针对寄主与病原菌的相互作用，我们提出了以下具体目标：1) 确定是否需要ORF2表达才能从 分枝杆菌酸合成酶体内合成邻苯二甲酸乙二醇酯和酚硫代乙醇酯 OrJ2基因突变及其对人类免疫功能的影响 霉菌基脂生物合成。如果发现有必要使用ORF2，我们将 阐明其参与霉菌基脂类合成的机制 并确定了ORF2的结构。2)阐明短链的本质 分枝杆菌酸合成酶基因的克隆和测序鉴定 活动站点域组织和子单元组成。3)击倒 结核分枝杆菌短链分枝杆菌酸合成酶基因的研究 并确定生化和功能后果。4)提纯 新发现的支链脂肪酸合成酶，鉴定产生的产物， 在基因组数据中寻找编码合成酶的基因的身份 Bank使用蛋白质序列，并确定生化和 敲除这一基因的功能后果。5)确定 结核分枝杆菌1类瘤样基因msl1和msl2的功能 它们在耻垢分枝杆菌中表达的产物的特征，以及通过 它们的生化和功能后果的测定 颠覆。6)确定2类(msl3、msl4、msl5)的功能 以及结核分枝杆菌基因组中的其他类似肿瘤的基因，通过对 它们的产物在耻垢分枝杆菌中表达，并通过测定 它们的破坏造成的生化和功能后果。7)确定 上述基因突变导致的特定脂类缺乏是否会影响 寄主-病原菌相互作用与致病力。结果将有助于识别 细胞壁脂类对发病至关重要，可能是合适的靶标 寻找新的抗分枝杆菌药物。

DESCRIPTION (Adapted from the applicant's abstract): Resurgence of tuberculosis and other mycobacterial infections associated with AIDS threaten human health world-wide. Emergence of drug resistant TB makes it critical to discover new drug targets. Mycobacterial cell walls contain a variety of lipids with many types of multiple methyl-branched fatty acids that are unique to pathogenic mycobacteria. These lipids are thought to play important roles in the ability of the pathogens to evade the defense reactions of the host. Biosynthesis of these unique lipids containing multiple-methyl branched cell wall lipids could offer ideal targets for new antimycobacterial drugs. To elucidate the mechanisms involved in the biosynthesis of these lipids and to test for their possible functions in host-pathogen interaction, we propose the following specific aims: 1) Determine whether ORF2 expression is required for mycocerosyl transfer from mycocerosic acid synthase to phthiocerol and phenolphthiocerol in vivo by disruption of the orJ2 gene and examination of the consequence on mycocerosyl lipid biosynthesis. If ORF2 is found to be necessary, we will elucidate the mechanism of its participation in mycocerosyl lipid synthesis and determine the structure of ORF2. 2) Elucidate the nature of short-chain mycocerosic acid synthase by cloning and sequencing the gene to identify the active site domain organization and subunit composition. 3) Knock-out the gene that encodes short-chain mycocerosic acid synthase in M. tuberculosis and determine the biochemical and functional consequences. 4) Purify a newly-found branched fatty acid synthase, identify the products generated, seek the identity of the gene that encodes the synthase in the genomic data bank using the protein sequence, and determine the biochemical and functional consequences of knocking-out this gene. 5) Determine the functions of Class 1 mas-like genes, msl1 and msl2, of M. tuberculosis by characterization of their products expressed in M. smegmatis, and by determination of the biochemical and functional consequences of their disruption. 6) Determine the functions of Class 2 (msl3, msl4, and msl5) and other mas-like genes in M. tuberculosis genome, by characterization of their products expressed in M. smegmatis, and by determination of the biochemical and functional consequences of their disruption. 7) Determine whether lack of specific lipids caused by the above gene-disruptions affects host-pathogen interaction and virulence. The results will help identify cell wall lipids critical for pathogenesis that might be suitable targets for new antimycobacterial drugs.