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

D-CLAP split-luciferin imaging shows that masking carboxylates as methyl esters increases cytosolic accumulation in luciferase-expressing Mycobacterium smegmatis, linked to higher antimycobacterial potency.

• Why it matters: Antibiotics must traverse the multilayered mycobacterial envelope to reach cytosolic targets, but cytosolic arrival is largely unmeasured, limiting structure–accumulation optimization amid rising nontuberculous infections and multidrug resistance.
• What they did: The authors adapted D-CLAP to luciferase-expressing Mycobacterium smegmatis and Mycobacterium avium, using disulfide-linked D-cysteine tags that release in the reducing cytosol to form D-luciferin and produce time-resolved luminescence.
• The result: D-CLAP enabled real-time cytosolic entry readouts for small-molecule antibiotics, polyarginine peptides, and griselimycin analogs, and a fluorescent polyarginine peptide associated with M. smegmatis in macrophage phagosomes.

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