Study on In Vitro Antibacterial Activity and Mechanism of Action of Artificially Designed Antimicrobial Peptide Amphilysin-K20

The problem of bacterial resistance is becoming increasingly severe, and the development of novel AMPs (antimicrobial peptides) is of great significance. This study aimed to rationally design an AMP with high efficacy and low toxicity, and explore its antibacterial mechanism. Based on the (XXYY)n template, an amphi philic α-helical antimicrobial peptide Amphilysin-K20 carrying eight positive charges was constructed through a synergistic optimization strategy. A series of methods including in vitro antibacterial experiments MIC (minimum inhibitory concentration), MBC (minimum bactericidal concentration), and time-kill curves, cytotoxicity assay, hemolysis test, electron microscopy observation, and molecular dynamics simulation were employed to systemati cally evaluate its activity and mechanism. The results showed that this peptide exhibits potent bactericidal activity against both Gram-positive and Gram-negative bacteria, with a MBC to MIC ratio of ≤2. Moreover, it achieves concentration-dependent rapid bactericidal effects within 150 min. Results from electron microscopy and molecular dynamics simulations revealed that Amphilysin-K20 exerts its rapid bactericidal action by disrupting the integrity of bacterial cell membranes. At a concentration of 16 μg/mL, it displayed low cytotoxicity toward HaCaT cells (cell viability>90%) and a hemolysis rate of only 1.39%, indicating excellent biosafety. In conclusion, this study success fully designed a novel antimicrobial peptide, Amphilysin-K20, through a rational “backbone-terminal” synergistic optimization strategy. Its membrane-targeted rapid bactericidal mechanism and favorable biocompatibility not only provide a theoretical basis for antimicrobial peptide design but also offer a promising candidate molecule for ad dressing the increasingly severe issue of bacterial resistance.

CSTR: 32200.14.cjcb.2026.02.0004