Photoactivatable Glycolipid Probes for Identifying Mycolate–Protein Interactions in Live Mycobacteria

Mycobacteria have
a distinctive glycolipid-rich outer membrane,
the mycomembrane, which is a critical target for tuberculosis drug
development. However, proteins that associate with the mycomembrane,
or that are involved in its metabolism and host interactions, are
not well-characterized. To facilitate the study of mycomembrane-related
proteins, we developed photoactivatable trehalose monomycolate analogues
that metabolically incorporate into the mycomembrane in live mycobacteria,
enabling in vivo photo-cross-linking and click-chemistry-mediated
analysis of mycolate-interacting proteins. When deployed in Mycobacterium smegmatis with quantitative proteomics, this
strategy enriched over 100 proteins, including the mycomembrane porin
(MspA), several proteins with known mycomembrane synthesis or remodeling
functions (CmrA, MmpL3, Ag85, Tdmh), and numerous candidate mycolate-interacting
proteins. Our approach is highly versatile, as it (i) enlists click
chemistry for flexible protein functionalization; (ii) in principle
can be applied to any mycobacterial species to identify endogenous
bacterial proteins or host proteins that interact with mycolates;
and (iii) can potentially be expanded to investigate protein interactions
with other mycobacterial lipids. This tool is expected to help elucidate
fundamental physiological and pathological processes related to the
mycomembrane and may reveal novel diagnostic and therapeutic targets.