Bacterial Aminopeptidase–Activated Peptide Prodrug Enables Species-Selective Targeting of Pseudomonas aeruginosa

Pseudomonas aeruginosa is an adaptable organism, frequently found in chronic infections, and for which antimicrobial resistance is a growing concern. Therefore, there is an urgent need for alternative therapeutic strategies. Cationic antimicrobial peptides (AMPs) offer potent bactericidal activity but suffer from limited selectivity and potential host toxicity. To enhance species-specific targeting, we designed two prodrug variants of the AMP D-Bac8C ^Leu2,5 – EEEE-D-Bac8C ^Leu2,5 and ELEG-D-Bac8C ^Leu2,5 — engineered for activation by the P. aeruginosa extracellular aminopeptidase PaAP. While both prodrug motifs effectively neutralized the positive charge of D-Bac8C ^Leu2,5 and prevented DNA–peptide complex formation, EEEE-D-Bac8C ^Leu2,5 showed negligible antimicrobial activity due to slow and incomplete activation. In contrast, ELEG-D-Bac8C ^Leu2,5 underwent rapid PaAP-mediated activation, restoring bactericidal activity in planktonic cultures and biofilms. PaAP contributed significantly to complete prodrug activation, particularly within biofilms, where the accumulation of partially activated intermediates correlated with biphasic killing kinetics. The prodrug showed reduced activity against other ESKAPEE pathogens, demonstrating selective activation by P. aeruginosa . Experiments selecting resistant bacteria revealed distinct mutations in lipopolysaccharide biosynthesis pathways for D-Bac8C ^Leu2,5 and the prodrug, with limited cross-resistance. These findings establish aminopeptidase-activated AMP prodrugs as a promising approach for species-selective antimicrobial therapy and highlight the feasibility of exploiting bacterial enzymes for controlled antimicrobial peptide activation.