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.