Lipid storage and mobilization in M. tuberculosis as antilatency drugs targets

描述（由申请人提供）：结核病（TB）是可预防死亡的主要原因。结核分枝杆菌（Mtb）在宿主体内以不复制的耐药状态潜伏数十年，并在人体免疫力减弱时重新激活的能力，是治疗和根除这种每年导致近200万人死亡的疾病的主要障碍。众所周知，脂肪酸是Mtb在宿主体内持续存在所需的主要能量来源。然而，脂肪酸的来源仍然未知。我们假设Mtb以三酰甘油（TG）和蜡酯（We）的形式储存能量，以成功地度过潜伏期。结核分枝杆菌储存和调动这些能量来源的步骤可能成为消灭潜伏结核分枝杆菌的新型抗潜伏药物的理想靶点。为了阐明这些步骤，我们建议：(1)生成tgs， fcr, fabp, fatp， cut和lip基因的基因破坏突变体，这些基因可能编码可能参与脂质储存和动员的酶，以成功通过潜伏期。(2)检测tgs、lip、cut和fcr基因在Mtb中的生化功能a)确定应激条件下tgs、lip、cut和fcr基因的破坏对Mtb中脂质积累的影响，从而诱导Mtb进入休眠状态b)阐明lip和cut突变体在动员储存的脂质的可能作用c)检测tgs、lip和cut基因在Mtb脂质代谢的酰基转移反应中的可能作用。(3) a)利用一种新的体外多重胁迫Mtb休眠培养模型，鉴定休眠发育过程中参与TG和WE积累的tgs和fcr基因。b)建立一种使用体外多重应激模型测试抗潜伏期候选药物的方法。（4) a)检测缺氧条件下THP-1源性巨噬细胞（TDM）中Mtb是否进入休眠状态b）检测负载脂质TDM中Mtb中积累的脂质体是否来源于宿主储存脂质，并鉴定参与脂质积累的Mtb基因c)通过研究脂质积累缺陷突变体是否不能产生Rif耐药来确定Mtb在TDM中积累脂质体是否与Rif耐药的发生有关。在两种体外休眠模型中均未表现出休眠特征的基因破坏突变体，将在兔结核潜伏期遏制/地塞米松再激活模型中测试其成功通过休眠和再激活的能力。它们还将提交给TARGET，在小鼠休眠的中空纤维人工肉芽肿模型中进行测试，以确定储存脂质积累是否参与该体内休眠模型。(5)在两种体外休眠模型中均未表现出休眠特征的基因破坏突变体，将在结核潜伏期的兔笼/地塞米松再激活模型中测试其成功通过休眠和再激活的能力。它们还将提交给TARGET，在小鼠休眠的中空纤维人工肉芽肿模型中进行测试，以确定储存脂质积累是否参与该体内休眠模型。预期的结果可能有助于确定能够消除潜伏结核分枝杆菌的新药的靶标。公共卫生相关性：结核病是可预防死亡的主要原因，每年造成200万人死亡。致病病原体可以保持潜伏状态，当人的免疫系统在生命中的某个时候被削弱时，病原体会被重新激活。世界三分之一的人口患有潜伏性结核病，目前尚无有效的药物治疗潜伏性结核病。这一建议是为了发现新的靶向药物，将有效对抗潜伏性结核病。这些药物是完全治愈和全球根除结核病所必需的。

DESCRIPTION (provided by applicant): Tuberculosis (TB) is a leading cause of preventable death. The ability of Mycobacterium tuberculosis (Mtb) to remain latent in a nonreplicating drug resistant state in the host for decades, to be reactivated when the immunity of the person weakens, is a major impediment to curing and eradicating this disease that causes nearly two million deaths annually. Fatty acids are known to be the major energy source required for the persistence of Mtb in the host. However, the source of the fatty acids remains unknown. We postulate that Mtb stores energy as triacylglycerol (TG) and wax esters (WE) to successfully go through latency. The steps Mtb uses to store and mobilize these energy sources could be ideal targets for novel antilatency drugs that will eliminate latent Mtb. To elucidate these steps we propose to: (1) Generate gene-disrupted mutants for tgs, fcr, fabp, fatp, cut and lip genes that may encode enzymes that could be involved in the storage and mobilization of lipids for successfully going through latency. (2) Examine the biochemical functions of tgs, lip, cut, and fcr genes in Mtb a) Determine the effect of disruption of tgs, lip, cut and fcr genes in the accumulation of lipids in Mtb under stress conditions that induce a dormancy-like state b) Elucidate the possible role of the lip and cut mutants on the mobilization of the stored lipids c) Examine the possible role of tgs, lip and cut genes in acyl transfer reactions in Mtb lipid metabolism. (3) a) Utilize a novel in vitro multiple stress Mtb culture model of dormancy to identify the tgs and fcr genes involved in the accumulation of TG and WE during development of dormancy. b) Develop a method to use the multiple stress in vitro model to test antilatency drug candidates. (4) a) Test whether Mtb goes into a dormant state in THP-1 derived macrophages (TDM) under hypoxia b) Determine whether the lipid bodies that accumulate in Mtb within lipid-loaded TDM originate from host storage lipids and identify Mtb genes involved in lipid accumulation c) Determine whether the lipid body accumulation by Mtb in TDM is associated with development of Rif resistance by investigating whether mutants defective in lipid accumulation fail to develop Rif resistance. Gene-disrupted mutants that fail to manifest dormancy traits in both in vitro models of dormancy will be tested for their ability to successfully go through dormancy and reactivation in a rabbit containment/dexamethasone reactivation model of TB latency. They will also be submitted to TARGET for testing in a hollow fiber artificial granuloma model of dormancy in mice and determine whether storage lipid accumulation is involved in this in vivo dormancy model. (5) Gene-disrupted mutants that fail to manifest dormancy traits in both in vitro models of dormancy will be tested for their ability to successfully go through dormancy and reactivation in a rabbit containment/dexamethasone reactivation model of TB latency. They will also be submitted to TARGET for testing in a hollow fiber artificial granuloma model of dormancy in mice and determine whether storage lipid accumulation is involved in this in vivo dormancy model. The expected results are likely to help identify targets for novel drugs that can eliminate latent Mtb. PUBLIC HEALTH RELEVANCE: Tuberculosis (TB) is a leading cause of preventable death causing two million deaths annually. The causative pathogen can stay in a latent state and get reactivated when the person's immune system is weakened some time during life. One third of the world population has latent TB and no currently available drug is effective against latent TB. This proposal is to discover targets for novel drugs that will be effective against latent TB. Such drugs are required to get complete cure and global eradication of TB.