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重大威胁。疾病预防控制中心已将耐多药结核分枝杆菌（MTB）菌株列为C类，列入可能用于生物恐怖主义的生物清单。迫切需要发现新的抗分枝杆菌药物靶点，以对抗这些威胁，特别是耐多药结核病。结核分枝杆菌基因组中脂质代谢基因异常丰富。越来越清楚的是，脂质代谢在结核病中起着关键作用。当宿主出现免疫缺陷时，在病原体发展为活动性结核病之前，处于休眠状态的病原体感染和存活几十年所需要的关键步骤可以成为新型抗结核病药物的理想靶点。我们推测tgs/wes基因（TG合成酶/蜡酯合成酶基因）和lip基因参与了毒力和病原体在休眠条件下的存活。我们将检验这个假设。1)阐明tgs/wes基因产物的生化功能。a)表征tgs/wes基因产物在大肠杆菌中表达的酶活性。b)确定破坏每个tgs/wes基因对脂质代谢的生化后果。2)确定tgs/wes基因破坏对宿主-病原体相互作用的影响；a)确定当结核分枝杆菌在培养中达到缺氧诱导的非复制状态时，是否可以检测到与诱导TG合成相关的任何分子变化；b)确定突变体在巨噬细胞中生长和触发细胞因子产生的能力是否改变；tgs/wes突变体在小鼠体内休眠并在宿主免疫功能低下时引起感染的能力。3)阐明lip基因的生化功能a)表达并表征在大肠杆菌中表达的lip基因产物的TG水解酶和硫酯酶活性。b)破坏每个唇基因并确定生化后果。4)确定唇部干扰物对宿主-病原体相互作用的影响，a)确定唇部基因干扰物在缺氧诱导的非复制状态下存活的能力，b)确定唇部基因干扰物对巨噬细胞生长和细胞因子产生的影响。c)确定唇基因破坏对免疫功能低下宿主的毒力、持久性以及休眠和再激活能力的影响。5)阐明已知毒力因子二真菌基硫代酚（dimycocerosylphthiocerol， DIM）的生物合成机制。鉴定对疾病至关重要的脂质代谢的独特步骤将允许寻找针对这些靶点的新药。

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.