Enhanced proteolytic stability and distinct mechanisms of a D-amino acid-modified antimicrobial peptide against Pseudomonas aeruginosa.

Antimicrobial peptides (AMPs) are promising alternatives to conventional antibiotics for the treatment of multidrug-resistant (MDR) pathogens; however, their clinical translation is limited by proteolytic degradation. We developed dPA-13, an all-D-enantiomer of the 13-mer AMP PA-13, to improve resistance. dPA-13 exhibited potent bactericidal activity against Pseudomonas aeruginosa, superior protease stability, and a favorable safety profile. Confocal imaging and comparative proteomics revealed distinct mechanistic differences: while PA-13 localized to the membrane, dPA-13 exhibited enhanced cytoplasmic translocation. Proteomic analysis showed dPA-13 triggers a comprehensive cellular response, upregulating oxidoreductases (e.g., catalase), DNA repair machinery, and metal-ion binding proteins, while downregulating metabolic enzymes, transport permeases, and virulence factors (PilT and flagellar systems). Molecular docking corroborated these shifts, identifying high binding affinities toward critical targets, notably PilT, stabilized by specific electrostatic and hydrogen-bonding motifs at Glu394 and His397. Furthermore, P. aeruginosa developed resistance to dPA-13 significantly slower than to ciprofloxacin. Collectively, dPA-13 is a proteolytically robust, dual-action antimicrobial that disrupts both membrane integrity and intracellular homeostasis, positioning it as a promising candidate for recalcitrant MDR infections.