SUMMARY 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. Mtb has two enzymes that can oxidize cholesterol and oxysterols in this way: 3-hydroxysteroid dehydrogenase (Hsd/Rv1106c) and cholesterol oxidase (ChoD/Rv3409c). These enzymes can convert the hydroxy residue at the third carbon position of cholesterol to a keto moiety. We hypothesized that cholestenone and 3-oxocholestenoic acid might be specific markers of TB infection. Indeed, in two geographically distinct cohorts, the level of these metabolites in sputum distinguished subjects with active TB from TB-negative individuals who presented with TB-like symptoms. In addition, their abundance correlated with the degree of smear positivity, a rough estimate of bacterial burden. Why these metabolites are elevated during TB and the impact that they have on infection are unknown. We hypothesize that Mtb Hsd and ChoD disrupt the repertoire of immune active oxysterols by converting them from 3-hydroxy to 3-oxo-derivatives, thereby interfering with the host immune response. In support of this idea, we found that mice infected with hsd Mtb and choD Mtb have altered inflammatory responses in the lungs compared to mice infected with WT Mtb. Here, using two mouse models of TB and a highly sensitive, well-established metabolomics pipeline that is optimized for oxysterol analysis, we will comprehensively profile oxysterols in the lungs, establish the role of Hsd, ChoD, and host enzymes in the generation of the 3-oxo metabolites, and assess the impact of the Mtb-induced metabolites on the host inflammatory response and disease outcomes. In addition, we propose that the shift in oxysterol metabolites creates a unique signature of TB disease that can be used as a diagnostic and treatment biomarker. We will comprehensively profile oxysterols from TB patients in Asia, Africa, and South America and determine whether 3-oxo-modified oxysterols decline in TB patients when they are on effective therapy. Thus, in both mice and humans, we will establish the profile of immune active oxysterols in the lungs and how they are modified by TB. Our studies will have an important impact on the TB field by elucidating fundamental mechanisms of pathogenesis and by advancing biomarker development, which has the potential to substantially improve clinical care. More broadly, our work will provide insight to the role of oxysterols in lung inflammation and immunity.

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 goal of this project is to determine whether Mtb infection alters host oxysterol metabolites, thereby modifying the host inflammatory response and promoting disease pathogenesis. These findings could lead to novel therapies and diagnostics for TB.