Split-luciferin Assay for Real-time Measurement of Cytosolic Molecular Accumulation in Live Mycobacteria

Antibiotics against nontuberculous mycobacteria must cross a multilayered cell envelope before reaching their cytosolic targets, yet how much of a given compound arrives there is largely unmeasured. This gap matters because nontuberculous mycobacterial infections are rising among immunocompromised individuals and those with structural lung disease, and intrinsic and acquired multidrug resistance creates an urgent need for new agents. Existing methods quantify whole-cell association without resolving subcellular localization, and none report accumulation in live mycobacteria, in real time. Here we adapt a split-luciferin approach, the D-cysteine-linked accumulation profiling (D-CLAP), to luciferase-expressing Mycobacterium smegmatis and Mycobacterium avium. Compounds bearing a disulfide-linked D-cysteine tag release the tag upon entry into the reducing cytosol, where it condenses with an intracellular cyanobenzothiazole to form D-luciferin and generate a time-resolved luminescent signal. We applied D-CLAP to small-molecule antibiotics, polyarginine peptides, and griselimycin analogs, and found that masking carboxylates as methyl esters increased both cytosolic accumulation and antimycobacterial potency. A fluorescent polyarginine peptide also associated with M. smegmatis residing within macrophage phagosomes. D-CLAP provides a real-time readout of cytosolic entry in live mycobacteria and a route to structure-accumulation relationships.