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

Tuberculosis causes over one million deaths annually and remains the leading cause of death from a single infectious agent. The emergence of multidrug-resistant Mycobacterium tuberculosis strains highlights the urgent need for new antibiotics, a pursuit hindered by the bacterium's complex cell envelope. As most anti-tuberculosis agents act on intracellular targets, assessing cytosolic drug accumulation is critical. Conventional approaches generally quantify whole-cell association without resolving subcellular localization. Moreover, no current method permits real-time monitoring of drug accumulation in live mycobacterial cells. Here, we present a split-luciferin-based assay to quantify molecular accumulation in mycobacteria. Using this approach, we quantified the cytosolic accumulation of diverse small-molecule antibiotics and polyarginine peptides conjugated via a disulfide-linked D-cysteine tag. We also show the localization of a polyarginine peptide inside of mycobacteria in infected macrophage cells, demonstrating that these peptides can cross multiple accumulation barriers. Our findings establish the first assay for real-time quantification of cytosolic molecular accumulation in live mycobacteria, addressing a longstanding methodological gap and enabling mechanistic insights into intracellular drug uptake.