SUMMARY (for Supplement) Mycobacterium tuberculosis (Mtb) causes one of the world's most deadly infections. How Mtb modulates the host immune response in order to establish infection, persist in the face of adaptive immunity, and elicit tissue pathology to transmit is not well understood. Mtb grows intracellularly in lipid-laden (foamy) macrophages and extracellularly within cholesterol-rich caseum of liquified granulomas. Mtb does not make cholesterol, but Mtb can degrade host cholesterol and use it as a carbon source. The host modifies cholesterol by enzymatically oxidizing it to a variety of derivatives, called oxysterols, which modulate the immune response. We found that two oxidized cholesterol metabolites, cholestenone and 3-oxocholestenoic acid, accumulate in Mtb-infected mouse lung, rabbit granulomas, and human sputum. The Mtb enzyme, 3-hydroxysteroid dehydrogenase (Hsd/Rv1106c), converts the hydroxy residue at the third carbon position of cholesterol to a keto moiety. The hypothesis underlying our parent grant is that Mtb Hsd disrupts the repertoire of cholesterol metabolites, including immune active oxysterols, by converting them from 3-hydroxy to 3-oxo-derivatives, thereby interfering with the host immune response. We found that in vitro and during macrophage infections Hsd is essential for converting cholesterol to its 3-oxo-derivative, cholestenone. However, how Hsd accesses host cholesterol is unclear. The previous literature assumed that cholesterol had to be imported into Mtb through the Mce4 cholesterol importer in order to undergo this oxidation step, which was thought to be a required first step in cholesterol degradation. However, our new preliminary data demonstrate that cholesterol does not have to be imported into Mtb to be oxidized, suggesting that cholestenone is generated outside of the bacteria, where it can impact host cell processes. How Hsd, which lacks a signal sequence, is being exported from Mtb and where it accesses host cholesterol is unknown. In this NIH Research Supplement to Promote Diversity in Health-Related Research, we will determine whether Hsd is in extracellular vesicles (EVs), establish where Hsd localizes within host cells, and examine the role of bacterial and host factors in cholesterol oxidation. These experiments support the overall objectives of the parent grant, while following up on an exciting new finding. The supplement will extend our work mechanistically and provide a high quality experimental plan to ensure Jalalah Muhammad's career development and future success.

PROJECT NARRATIVE Mycobacterium tuberculosis (Mtb)- the causative agent of tuberculosis (TB)- is able to establish infection in the lungs, persist in humans for years, and kill more people than any other bacteria. The scientific goal of this supplement is to determine how the Mtb enzyme, 3-hydroxysteroid dehydrogenase, accesses host cholesterol to modify the host inflammatory response and promote disease pathogenesis. These findings could lead to novel therapies and diagnostics for TB.