Antimicrobial peptide mechanism of action on S. aureus membranes determined by in vivo solid-state NMR

Staphylococcus aureus (S. aureus) is a Gram-positive pathogenic bacterium and a major cause of nosocomial infections. Between 20 % and 50 % of S. aureus strains are resistant to a wide range of antibiotics. DMS-DA6-NH₂ (DA6) is a novel antimicrobial peptide (AMP) that exhibits high efficacy against various bacterial strains, particularly S. aureus, by disrupting its membrane through an as-yet-unknown mechanism. We employed in vivo²H solid state Nuclear Magnetic Resonance (NMR) to investigate the mode of action of AMPs on deuterated bacteria. This technique provides insights into membrane order and its changes with increasing AMP concentration. Our results enabled us to compare the mechanism of DA6 with those of AMPs with established modes of action. We found that DA6 induces pore formation in the membrane of S. aureus. This protocol serves as a template for determining the mechanisms of action of other peptides, an essential step for developing and patenting such drugs for the treatment of human diseases.