Cholesterol oxidation products in TB pathogenesis and as biomarkers of disease

SUMMARY Mycobacterium tuberculosis (Mtb) causes tuberculosis (TB), killing more people than any other pathogen. Mtb is able to subvert innate immunity, impairing the antimicrobial function of macrophages. Mtb induces the formation of lipid-droplet-filled or “foamy” macrophages, and the bacilli themselves can be found in close

approximation to intracellular lipid droplets, which are thought to serve as a source of nutrients in the form of cholesterol esters and fatty acids for the bacilli. When we performed global metabolic profiling of Mtb infected macrophages, we found that the cholesterol metabolite, cholestenone, was the second most differentially

expressed metabolite between infected and uninfected cells. The presence of cholestenone reflects the activity of both the host cell, which is the source of cholesterol, and a bacterial cholesterol oxidase. Mtb is known to oxidize cholesterol, which is thought to be the first step in a degradative pathway that enables Mtb to utilize

cholesterol as a carbon source. We hypothesize that oxidized cholesterol is more than an intermediate in a degradative pathway; we propose that it plays a role in virulence by interfering with antimicrobial function of macrophages. Further, we propose that cholestenone will be a useful biomarker of TB disease burden. Here,

we will establish whether bacterial-derived cholesterol metabolites contribute to immune evasion and whether they can be used for diagnostics to evaluate disease burden. Cholestenone is known to disrupt cellular membranes, interfering with lipid rafts. The assembly and activity of the NADPH oxidase is sensitive to

membrane lipid composition and raft integrity. We will establish whether cholestenone impairs NADPH oxidase assembly, lysosomal trafficking, and intracellular growth using protocols that are well-established in the lab. We will monitor the distribution of cholestenone in infected macrophages using mass-spectrometry compatible

fractionation strategies and novel diazirine alkyne probes to visualize its subcellular localization. Although there is consensus that Mtb can utilize cholesterol as a carbon source, there is discrepancy as to the identity of the cholesterol oxidase. We will establish whether 3β-hydroxysteroid dehydrogenase and ChoD, putative cholesterol

oxidases, and Mce4, a cholesterol uptake system, are required for cholesterol oxidation in vitro and during macrophage infection. Finally, we will determine whether cholestenone abundance distinguishes sputum from TB-infected individuals from uninfected controls. Currently, there are no biomarkers that predict disease burden

or response to therapy. Thus, a biomarker that reflects bacterial burden and response to therapy would have a major impact on patient care. Overall, our studies have the potential to make an important impact on one of the most formidable pathogens by (1) elucidating mechanisms of Mtb pathogenesis, (2) clarifying an important

metabolic pathway, and (3) leading to the development of much needed, novel diagnostics.