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.