Development of a Lipidomics Platform to Discover Enzymatic Activities of Mycobacterial Proteins of Unknown Function



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Mycobacterium tuberculosis (Mtb), a facultative intracellular pathogen, is responsible for 10 million new cases of tuberculosis and 1.5 million deaths annually. Mtb infection can be acute but also lifelong. Indeed, despite the devastating global impact on health, how Mtb is able to adapt to the human body and survive for years is unknown. To better understand the physiology of Mtb infection, exploring the functions of individual proteins produced by Mtb is critical. Less than half of the proteins encoded in the M. tuberculosis genome have been fully characterized either directly through experimentation or indirectly through annotation. Many of these unknown genes likely encode proteins important for pathogenesis and adaptation to host responses. I created a novel method to facilitate the identification of novel substrates and products using direct infusion tandem mass spectrometry (DI-MS/MS) analysis of nonpolar metabolite fluctuations after transient and rapid protein overexpression. I studied 24 proteins using this lipidomic method. Of these, several demonstrate unique patterns of metabolites secondary to rapid induction of protein expression, and work towards identifying their specific enzymatic activities is ongoing. I selected one protein, Cor (Rv1829), for further characterization as it was recently shown to be fundamental to carbon monoxide (CO) resistance and pathogenesis of Mtb during host infection. To study Cor in vitro, I purified recombinant Cor from E. coli and used activity based metabolic profiling (ABMP) to measure changes in mycobacterial metabolites exposed to Cor. In the ABMP assay, exposure of mycobacterial metabolites to Cor led to consumption of acetyl phosphate and accumulation of phosphatidic acid (PA). I developed direct enzymatic assays for Cor activity, and confirmed the consumption of acetyl phosphate. To assess substrate binding, I used isothermal titration calorimetry (ITC) and demonstrated binding of Cor to acetyl phosphate. Additionally, Cor interacted directly with cardiolipin (CL), phosphatidylglycerol (PG), phosphatidylserine (PS), phosphatidylinositol (PI), and sulfatide on membrane lipid strips. I overexpressed Cor in M. smegmatis and Mtb and lipids were extracted and analyzed through DI-MS/MS to assess changes in lipid composition. I combined in vivo lipidomics and in vitro ABMP to biochemically characterize Cor and found that Cor is interacting with bacterial lipids. In vivo lipidomic analysis revealed an accumulation of PA and PG by 3 hours. Our findings suggest that Mtb Cor is interacting with lipids involved in the phospholipid biosynthesis pathway and modifying bacterial lipid composition by accumulating PG. These experiments provide mechanistic insight into the enzymatic function of Cor. Furthermore, the metabolomics and lipidomics approaches developed in this study can be broadly applied to study proteins of unknown function from other organisms.

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