Search for novel TB Drug Targets in Lipid Metabolism

寻找脂质代谢中的新型结核病药物靶点

• 批准号: 6723620
• 负责人: PAPPACHAN KOLATTUKUDY KOLATTUKUDY
• 金额: $ 35.88万
• 依托单位: UNIVERSITY OF CENTRAL FLORIDA
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-01-15 至 2008-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6723620
• 关键词: Escherichia coli; Mycobacterium smegmatis; Mycobacterium tuberculosis; antitubercular agents; bacteria infection mechanism; disease /disorder prevention /control; drug resistance; emerging infectious disease; enzyme activity; enzyme complex; fatty acids; gene expression; gene interaction; gene mutation; host organism interaction; laboratory mouse; lipid metabolism; molecular cloning; polyketide synthase; polymerase chain reaction; protein structure function; thin layer chromatography; tuberculosis; virulence

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.

描述（由申请人提供）：结核病 (TB) 是可预防死亡的主要原因，每年导致超过 200 万人死亡。多重耐药（MDR）结核病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) 菌株归为 C 类，属于可能用于生物恐怖主义的生物体列表。 对抗这些威胁，特别是耐多药结核病，迫切需要发现新型抗分枝杆菌药物靶点。 MTB 基因组中含有异常丰富的脂质代谢基因。人们越来越清楚，脂质代谢在结核病中发挥着关键作用。当宿主免疫缺陷时，病原体在发展为活动性结核病之前数十年处于休眠状态，感染和存活所需的关键步骤是新型抗结核药物的理想靶标。我们假设 tgs/wes 基因（TG 合酶/蜡酯合酶基因）和 lip 基因与病原体在休眠条件下的毒力和存活有关。我们将检验这个假设。 1）阐明tgs/wes基因产物的生化功能。 a) 表征大肠杆菌中表达的 tgs/wes 基因产物的酶活性。 b) 确定破坏每个 tgs/wes 基因对脂质代谢的生化影响。 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 MTB 在培养物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) 确定突变体的毒力、持久性和能力 tgs/wes 突变体在小鼠体内进入休眠状态，并在宿主免疫功能低下时引起感染。 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) 破坏每个嘴唇基因并确定生化后果。 4) 确定唇基因破坏剂对宿主-病原体相互作用的影响，a) 确定唇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) 确定唇基因破坏对巨噬细胞生长和细胞因子产生的影响。 c) 确定唇基因破坏对毒力、持久性以及在免疫受损宿主中经历休眠和重新激活的能力的影响。 5) 阐明涉及已知毒力因子 dimycocerosylphthiocerol (DIM) 生产的生物合成机制。鉴定对该疾病至关重要的脂质代谢的独特步骤将有助于寻找针对这些靶点的新药物。