Identification and Biochemical Characterization of Tetrahydrolipstatin Targets In M. Bovis BCG At Different Metabolic States

Abstract

Mycobacteria, like other prokaryotic species, are able to accumulate large amounts of neutral lipids such as triacylglycerol (TAG) containing structures called lipid droplets (LDs). A growing body of evidence indicates the importance of the roles played by LDs in the pathogenesis of mycobacterial diseases. In particular, recent reports have shown that tubercular bacilli in lung granulomas are enriched in LDs. These TAG deposits are consumed when dormant bacilli are reactivated, suggesting that lipid storage probably contributes to mycobacterial survival during the latent and/or reactivation phase. The TAG degradation process catalyzed by lipolytic enzymes may release free fatty acids, which can be utilized as a carbon source during growth and infection processes. Several studies have explored the possibility of utilizing inhibitors to target these lipid-catabolizing enzymes for the treatment of latent tuberculosis. In addition to the lipolytic enzymes, there is also an interest in studying the non-lipolytic esterases, which aid mycobacterial survival under stress conditions by detoxifying toxic metabolites and drugs. Members of this family of enzymes are also considered to be opportunistic enzymes that generate energy from soluble TAGs and free-esters. Our group has previously demonstrated that tetrahydrolipstatin (THL), an irreversible inhibitor of serine esterases, attenuates regrowth of dormant mycobacteria by preventing TAG breakdown. To better understand THL activity and its pharmacological implications in vivo, we used a novel chemical proteomic strategy to identify specific mycobacterial targets and to validate some of their biological activities. We report that THL targets the a/?-hydrolase family proteins, including the `Lip family? of enzymes and identified BCG2241c, lipD, lipM, lipN, Ag85c, lipI, lipH, lipW, BCG3408, BCG1252 and tesA as high confidence THL targets. THL is a lipase inhibitor and lipases are hypothesized to be up-regulated during mycobacteria regrowth from non-replicating persistent (NRP) bacilli. Therefore, to obtain a functional insight into mycobacterial esterases and/or lipases in mycobacterial NRP, we extended our chemical-proteomic strategy to other disease-relevant mycobacterial metabolic states, specifically NRP and regrowth from NRP. Interestingly, we observed that down-regulation of certain THL targets in the NRP phase, and the expression pattern was reversed during the subsequent regrowth. We further show that regulation of these genes occur at the proteomic level, maybe by post-translational modification mediated translocation, degradation or inactivation. Finally, among the high confidence THL targets, we selectively characterized lipH, a protein whose function was previously poorly defined, as an esterase with a preference towards short-chain free esters, whose non-lipolytic activity is inhibited by THL. A detailed quantitative time-course study further validated lipH as one of the THL targets down-regulated during NRP and revived within a day of regrowth. In addition, we propose that lipH might provide carbon source to energy-deficient mycobacteria resuscitating from NRP by acting on free-esters and may serve as a potential new drug target specifically targeting mycobacteria re-emerging from a NRP state.