Mtb-Catalyzed Cholesterol Oxidation as Virulence Factor

描述（由申请人提供）：结核病是一种机会性感染的个人与艾滋病毒/艾滋病，估计感染30%的世界人口。世界卫生组织估计，每年有200万人死于结核病。对一线结核病药物利福平和异烟肼的耐药性正在出现，导致人口结核病负担增加。显然，需要采取新的办法来对付这种致命的多重耐药生物体（MDR-TB）的出现。有许多报道表明分枝杆菌可氧化胆固醇，并且氧化活性是可诱导的。重要的是，胆固醇氧化与致病性有关。负责这种活性的酶包含抗TB药物通常不靶向的途径，并且它们的抑制可能导致在对抗感染中改善宿主反应。然而，负责该活性的蛋白质从未被分离出来，致病机制也知之甚少。所提出的实验旨在鉴定结核分枝杆菌中负责胆固醇氧化的基因和表征蛋白质。假设胆固醇氧化活性代表了抗MDR-TB药物开发的新靶点，因为1）胆固醇氧化对毒力至关重要，2）这些酶在人体中不存在。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，对M.结核病基因Rv 3409 c（choD）和Rv 1106 c将被表达和纯化。将表征其动力学活性和底物特异性。第二，对M.将测定在胆固醇存在和不存在下生长的肺结核，以确定胆固醇是否是Rv 3409 c和Rv 1106 c的调节剂，并确定胆固醇活化途径中存在哪些其他基因产物，从而确定氧化。此外，将纯化负责胆固醇氧化活性的天然蛋白质，以确定诱导的活性是否来源于预测的基因产物，并鉴定翻译后修饰。

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.