Comparative mode of action of antimicrobial peptide melimine and its derivative Mel4 against Pseudomonas aeruginosa

Melimine and Mel4 are chimeric cationic peptides with broad-spectrum antimicrobial activity, and highly biocompatible with animal-model and human clinical trials. The current study examined the mechanism of action of these two antimicrobial peptides against P. aeruginosa with a series of investigations such as Endotoxin neutralization, cytoplasmic membrane disruption using DiSC(3)-5 and Sytox green stains, and Syto-9 and PI dyes using flow cytometry. Release of ATP and DNA/RNA were determined using ATP luminescence and increase in OD260nm. Whereas, bacteriolytic ability of peptides was determined by decrease in OD620nm. Both the peptides efficiently neutralized LPS, suggesting their role in the anti-pseudomonas and LPS binding activities. Depolarization of cytoplasmic membrane by melimine and Mel4 released 75% and 36% ATP within two minutes (P<0.001) respectively. Membrane permeabilization started within 5 minutes of exposure to the peptides, with simultaneous release of DNA/RNA. Flow cytometry demonstrated 52% and 18% bacteria were PI stained after 30 minutes respectively. Overall, melimine showed higher capacity of membrane disruption, and cell lysis compared to Mel4 (P<0.001). Findings of this study has been summarized as a timeline of bactericidal activities, suggesting that the peptides permeabilised P. aeruginosa within 5 minutes, started lysis within 2 hours of exposure leading to rapid death.