Mtb-Catalyzed Cholesterol Oxidation as Virulence Factor

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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.