Bioorthogonal metabolic labeling of the virulence factor phenolic glycolipid in mycobacteria

ABSTRACT Surface lipids on pathogenic mycobacteria modulate infection outcomes by regulating host immune responses. Phenolic glycolipid (PGL) is a host-modulating surface lipid that varies among clinical Mycobacterium tuberculosis strains. PGL is also found in Mycobacterium marinum where it promotes infection of zebrafish through effects on the innate immune system. Given the important role this lipid plays in the host-pathogen relationship, tools for profiling its abundance, spatial distribution, and dynamics are needed. Here we report a strategy for imaging PGL in live mycobacteria using bioorthogonal metabolic labeling. We functionalized the PGL precursor p -hydroxybenzoic acid ( p HB) with an azide group (3-azido p HB). When fed to mycobacteria, 3-azido p HB was incorporated into the cell surface, which could then be visualized via bioorthogonal conjugation of a fluorescent probe. We confirmed that 3-azido p HB incorporates into PGL using mass spectrometry methods and demonstrated selectivity for PGL-producing Mycobacterium marinum and Mycobacterium tuberculosis strains. Finally, we applied this metabolic labeling strategy to study the dynamics of PGL within the mycobacterial membrane. This new tool enables visualization of PGL which may facilitate studies of mycobacterial pathogenesis.