Bioorthogonal Metabolic Labeling of the Virulence Factor Phenolic Glycolipid in Mycobacteria.

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 strains. PGL is also found in , 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 -hydroxybenzoic acid (HB) with an azide group (3-azido HB). When fed to mycobacteria, 3-azido HB was incorporated into the cell surface, which could then be visualized the bioorthogonal conjugation of a fluorescent probe. We confirmed that 3-azido HB incorporates into PGL using mass spectrometry methods and demonstrated selectivity for PGL-producing and 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 that may facilitate studies of mycobacterial pathogenesis.