New test reveals drug entry into tuberculosis bacteria in real time

Drug-resistant strains of tuberculosis and related mycobacteria highlight the urgent need for new antibiotics, but progress is slowed by the bacterium’s complex cell envelope. Most anti-tuberculosis agents must reach targets inside the cell to work, so knowing whether a candidate drug actually gets into the cytosol of the bacterium is critical. Until now, common laboratory methods have generally measured how much of a compound associates with whole cells without resolving where the compound sits inside the cell, and no method allowed researchers to watch cytosolic accumulation as it happens in live mycobacterial cells. To address this gap, Marcos M. Pires and colleagues developed a split-luciferin-based assay designed to report the presence of molecules inside the cytosol of live mycobacteria in real time. The new approach was built specifically to overcome the limitations of whole-cell association assays and to provide a direct, time-resolved readout of whether compounds cross the multiple barriers that separate the outside world from an intracellular target.

The core tool reported is a split-luciferin-based assay that produces a measurable signal when a tagged molecule accumulates in the cytosol. Using this approach, the team quantified the cytosolic accumulation of diverse small-molecule antibiotics and of polyarginine peptides that were conjugated via a disulfide-linked D-cysteine tag. The assay allowed measurement in live mycobacteria and provided a real-time view of accumulation behavior rather than a single endpoint measurement. The researchers also demonstrated localization of a polyarginine peptide inside of mycobacteria in infected macrophage cells, showing that these peptides can cross multiple accumulation barriers encountered in an infection setting. Together, these results establish the assay as a practical way to compare how different chemical classes and conjugation strategies influence access to the mycobacterial cytosol.

This work fills a longstanding methodological gap by delivering the first assay capable of real-time quantification of cytosolic molecular accumulation in live mycobacteria. That capability opens up new mechanistic insights into intracellular drug uptake: researchers can now observe how quickly and how much different compounds penetrate the cell, and how modifications such as peptide conjugation and disulfide-linked D-cysteine tags affect delivery to intracellular targets. In practical terms, the split-luciferin-based assay could help prioritize compounds during antibiotic discovery, guide medicinal chemistry to improve cytosolic access, and refine understanding of how bacteria and host cells influence drug distribution. By enabling measurements inside infected macrophage cells, the method also offers a route to study compound behavior in conditions that better mimic real infections, which may accelerate development of treatments that actually reach their intended targets inside mycobacteria.