Search for novel TB Drug Targets in Lipid Metabolism

描述（由申请人提供）：结核病（TB）是可预防死亡的主要原因，每年造成200多万人死亡。耐多药结核病的自然传播是对公共卫生的主要威胁。CDC已将M.结核病（MTB）在C类，在生物恐怖主义的潜在用途的生物体清单。 发现新的抗分枝杆菌药物靶点是对抗这些威胁的关键，特别是耐多药结核病。 MTB基因组中异常丰富的脂质代谢基因。越来越清楚的是，脂质代谢在结核病中起着关键作用。当宿主变得免疫缺陷时，在病原体发展为活动性TB之前，病原体在休眠状态下感染和存活数十年所独特需要的关键步骤可以是新型抗TB药物的理想靶标。我们假设，tgs/wes基因（TG合酶/蜡酯合酶基因）和唇基因参与的毒力和在休眠条件下的病原体的生存。我们将测试这个假设。1)阐明tgs/wes基因产物的生化功能。a）鉴定在E.杆菌B）确定破坏每个tgs/wes基因对脂质代谢的生物化学后果。2)确定tgs/wes基因破坏对宿主-病原体相互作用的后果，a）确定当MT B在培养物B中达到缺氧诱导的非复制状态时，是否可以检测到与TG合成诱导相关的任何分子变化，b）确定突变体是否具有改变的在巨噬细胞中生长和触发细胞因子产生的能力，c）确定突变体的毒力、持久性，以及当宿主免疫受损时，TGS/WES突变体在小鼠中进入休眠并引起感染的能力。3)a）在大肠杆菌中表达并鉴定lip基因产物的TG水解酶和硫酯酶活性;杆菌B）破坏每个lip基因并确定生化后果。4)确定lip破坏剂对宿主-病原体相互作用的影响，a）确定lip基因破坏剂在培养物中缺氧诱导的非复制状态下存活的能力，B）确定lip基因破坏对巨噬细胞生长和细胞因子产生的影响。 c）确定lip基因破坏对毒力、持久性以及在免疫受损宿主中经历休眠和再活化的能力的影响。5)阐明参与生产的二分枝蜡基苯硫代蜡醇（DIM），一个已知的毒力因子的生物合成机制。确定脂质代谢中对疾病至关重要的独特步骤将允许寻找针对这些目标的新药。

DESCRIPTION (provided by applicant): Tuberculosis (TB) is a leading cause of preventable deaths accounting for over two million deaths per year. Natural spread of multiple drug resistant (MDR) TB is a major threat to public health. CDC has classified MDR strains of M. tuberculosis (MTB) in Class C, within the list of organisms with potential use in bioterrorism. Discovery of novel anti-mycobacterial drug targets is critically needed to combat these threats, especially the MDR TB. The MTB genome is unusually rich in genes for lipid metabolism. It is becoming increasingly clear that lipid metabolism plays critical roles in TB. The critical steps, that are uniquely required for infection and survival of the pathogens in a dormant state for decades before the pathogens develops active TB when the host becomes immunodeficient, can be ideal targets for novel anti-TB drugs. We postulate that tgs/wes genes (TG synthase/wax ester synthase genes) and lip genes are involved in virulence and in the survival of the pathogen under dormant conditions. We will test this hypothesis. 1) Elucidate the biochemical functions of tgs/wes gene products. a) Characterize the enzymatic activities of the tgs/wes gene products expressed in E. coli. b) Determine the biochemical consequences of disrupting each tgs/wes gene on lipid metabolism. 2) Determine the consequence of tgs/wes gene disruption on host-pathogen interactions, a) Determine whether any molecular changes relevant to induction of TG synthesis can be detected as MTB reaches the hypoxia-induced nonreplicating state in culture b) Determine whether mutants have altered ability to grow in macrophages and trigger cytokine production, c) Determine the virulence, persistence, and the ability of the tgs/wes mutants to go into dormancy in mice and cause infection when the host is immunocompromised. 3) Elucidate the biochemical functions of the lip genes a) Express and characterize the TG hydrolase and thioesterase activities of lip gene products expressed in E. coli. b) Disrupt each lip gene and determine the biochemical consequences. 4) Determine the effect of lip disruptants on host-pathogen interaction, a) Determine the ability of the lip gene disruptants to survive the hypoxia-induced nonreplicating state in culture, b) Determine the effects of lip gene disruption on growth in macrophages and cytokine production. c) Determine the effect of lip gene disruption on virulence, persistence and the ability to undergo dormancy and reactivation in immunocompromised host. 5) Elucidate the biosynthetic mechanisms involved in the production of dimycocerosylphthiocerol (DIM), a known virulence factor. Identification of the unique steps in lipid metabolism critical for the disease will allow a search for novel drugs directed at these targets.