Mapping of Mycobacterial Enzymes Involved in Triacylglycerol Accumulation as Intrabacterial Lipid Inclusions Using Activity-Based Multitarget Inhibitor Probes

Lipids play a critical role in the physiology, life cycle, and pathogenicity of mycobacteria. They largely participate in host-pathogen interactions and fulfill important functions ranging from cell wall biosynthesis/maintenance, bacterial growth dynamics, and long-term persistence. In that context, triacylglycerol, a specific subtype of neutral lipid, is stored as intrabacterial lipid inclusions (ILI), which have been described as important structures for long-term survival and persistence within the host. However, the enzymes involved in ILI formation and degradation remain largely undefined. Using bio-orthogonal click-chemistry activity-based protein profiling (CC-ABPP) of newly synthesized oxadiazolone (OX), cyclophostin, and cyclipostins (CyC) probes, we report the direct capture of target proteins in Mycobacterium abscessus growing under carbon excess and nitrogen-deprived in vitro conditions that promote triacylglycerol production and ILI formation. This approach led to the identification of a set of 65 enzymes potentially involved in global processes related to ILI anabolism. Among these, the long-chain-fatty-acid--CoA ligase MAB_1978c/FadD15 has been validated as a pivotal enzyme that colocalizes on ILI and as a major contributor in ILI formation in M. abscessus.