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(MDR)的自然传播是对公众健康的主要威胁。CDC已将耐多药结核分枝杆菌(MTB)菌株归类为C类，列入可能用于生物恐怖主义的生物体名单。为了对抗这些威胁，尤其是耐多药结核病，迫切需要发现新的抗分枝杆菌药物靶点。结核分枝杆菌基因组中含有异常丰富的脂类代谢基因。越来越清楚的是，脂代谢在结核病中起着关键作用。这些关键步骤是病原体在宿主免疫缺陷时发展为活动性结核病之前，在休眠状态下感染和生存所唯一需要的关键步骤，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基因参与了病原菌在休眠条件下的毒力和生存。我们将检验这一假设。1)阐明TGS/WES基因产物的生化功能。A)鉴定在大肠杆菌中表达的TGS/WES基因产物的酶活性。B)确定干扰每个TGS/WES基因对脂类代谢的生化后果。2)确定TGS/WES基因突变对宿主-病原体相互作用的影响，a)确定当MTB达到低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)确定突变体是否改变了在巨噬细胞中生长并触发细胞因子产生的能力；c)确定TGS/Wes突变体的毒力、持久性以及当宿主免疫受损时TGS/Wes突变体进入休眠并导致感染的能力。3)阐明LIP基因a)表达的生化功能，并对表达产物的甘油三酯水解酶和硫代酯酶活性进行鉴定。B)干扰每个LIP基因并确定其生化后果。4)确定LIP基因干扰物对宿主-病原体相互作用的影响，a)确定LIP基因干扰物在低氧诱导的非复制状态下存活的能力，b)确定LIP基因干扰对巨噬细胞生长和细胞因子产生的影响。C)确定LIP基因中断对免疫受损宿主的毒力、持久性以及经历休眠和重新激活的能力的影响。5)阐明已知的毒力因子二霉蜡基硫代葡萄糖醇(DIM)的生物合成机制。识别脂代谢中对疾病至关重要的独特步骤将使人们能够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.