The study shows that the natural antimicrobial peptide LL‑37 can get inside human immune cells (macrophages), find the compartments where TB bacteria hide, and break the bacteria’s wall, killing them. This was visualized using a super‑high‑resolution microscope, confirming that LL‑37 works inside cells, not just outside.

The antimicrobial peptide LL-37 inhibits the growth of the major human pathogen <i>Mycobacterium tuberculosis</i> (<i>Mtb</i>), but the mechanism of the peptide-pathogen interaction inside human macrophages remains unclear. Super-resolution imaging techniques provide a novel opportunity to visualize these interactions on a molecular level. Here, we adapt the super-resolution technique of stimulated emission depletion (STED) microscopy to study the uptake, intracellular localization and interaction of LL-37 with macrophages and virulent <i>Mtb</i>. We demonstrate that LL-37 is internalized by both uninfected and <i>Mtb</i> infected primary human macrophages. The peptide localizes in the membrane of early endosomes and lysosomes, the compartment in which mycobacteria reside. Functionally, LL-37 disrupts the cell wall of intra- and extracellular <i>Mtb</i>, resulting in the killing of the pathogen. In conclusion, we introduce STED microscopy as an innovative and informative tool for studying host-pathogen-peptide interactions, clearly extending the possibilities of conventional confocal microscopy.