Mtb-Catalyzed Cholesterol Oxidation as Virulence Factor

描述（由申请人提供）：结核病是艾滋病毒/艾滋病患者的机会性感染，估计感染世界人口的30%。世界卫生组织估计，每年有200万人死于肺结核。对一线结核病药物利福平和异烟肼的耐药性正在出现，并导致人口结核病负担增加。显然，需要新的方法来对抗这种致命的多药耐药菌（MDR-TB）的出现。有许多报道称分枝杆菌氧化胆固醇，并且氧化活性是诱导的。重要的是，胆固醇氧化与致病性有关。负责这一活性的酶包括一个通常不是抗结核药物靶向的途径，它们的抑制可能导致改善宿主对抗感染的反应。然而，负责活性的蛋白质从未被分离出来，其致病性机制也知之甚少。提出的实验旨在鉴定基因和表征负责结核分枝杆菌胆固醇氧化的蛋白质或蛋白质。假设胆固醇氧化活性代表了抗耐多药结核病药物开发的新目标，因为1)胆固醇氧化对毒力至关重要，2)这些酶在人类中不存在。这项工作的目的是阐明胆固醇氧化在宿主细胞感染中的作用。首先，表达并纯化结核分枝杆菌基因Rv3409c （choD）和Rv1106c的蛋白产物。它们的动力学活性和底物特异性将被表征。其次，将确定在存在和不存在胆固醇的情况下生长的结核分枝杆菌的差异转录谱，以确定胆固醇是否是Rv3409c和Rv1106c的调节剂，并确定在胆固醇激活和氧化的途径中存在哪些其他基因产物。此外，负责胆固醇氧化活性的天然蛋白将被纯化，以确定诱导活性是否来自预测的基因产物，并确定翻译后修饰。

DESCRIPTION (provided by applicant): Tuberculosis is an opportunistic infection in individuals with HIV-AIDS that is estimated to infect 30% of the world's population. The World Health Organization estimates that 2 million people die every year from tuberculosis. Drug resistance to front-line TB drugs rifampicin and isoniazid is emerging and leads to an increased TB burden in the population. It is clear that new approaches are required to combat the emergence of this virulent multi-drug resistant organism (MDR-TB). There are many reports that mycobacteria oxidize cholesterol, and that the oxidation activity is inducible. Importantly, cholesterol oxidation has been linked to pathogenicity. The enzymes responsible for this activity comprise a pathway that is not typically targeted by anti-TB Pharmaceuticals, and their inhibition could lead to improved host response in combating infection. However, the protein(s) responsible for the activity has (have) never been isolated, and the mechanism of pathogenicity is poorly understood. The proposed experiments are aimed at identifying the gene and characterizing the protein or proteins responsible for cholesterol oxidation in Mycobacterium tuberculosis. The hypothesis is that cholesterol oxidation activity represents a new target for anti-MDR-TB pharmaceutical development because 1) cholesterol oxidation is essential for virulence and 2) these enzymes are absent in humans. The aim of this work is to elucidate the role of cholesterol oxidation in host cell infection. First, the protein products of M. tuberculosis genes Rv3409c (choD) and Rv1106c will be expressed and purified. Their kinetic activity and substrate specificity will be characterized. Second, the differential transcription profile of M. tuberculosis grown in the presence and absence of cholesterol will be determined to determine if cholesterol is the regulator of Rv3409c and Rv1106c and to determine what other gene products are present in the pathway of cholesterol activation, and hence, oxidation. In addition, the native protein responsible for cholesterol oxidation activity will be purified to determine if the induced activity derives from the predicted gene products, and to identify post-translational modifications.