Antibacterial mechanism and computer simulation analysis of a novel antimicrobial peptide inducing membrane disintegration in Gram-positive bacteria

LAB-4, a novel antimicrobial peptide screened from the microbiome of Shanxi aged vinegar, has been shown to inhibit Gram-positive bacterial infections, but its antibacterial mechanism remains unclear. This study aimed to elucidate the antibacterial mechanism of LAB-4 against Gram-positive bacteria at the cellular and molecular levels. A series of in vitro assays (including antibacterial activity, safety, stability, membrane integrity, and membrane permeability analyses) and computational simulation techniques (molecular docking and molecular dynamics simulations) were employed to systematically investigate the antibacterial efficacy and mechanism of LAB-4. The results showed that LAB-4 exhibited potent antimicrobial activity, rapid bactericidal effects, minimal hemolytic activity, no cytotoxicity, and high stability under various adverse conditions. In vitro assays revealed that LAB-4 inhibited bacterial growth in a concentration-dependent manner, primarily causing irreversible membrane damage in Gram-positive bacteria by disrupting membrane integrity, increasing membrane permeability, and inducing intracellular content leakage. Computational simulations indicated that LAB-4 initially bound to peptidoglycan, after which its positively charged amino acid residues (primarily Arg, His, and Lys) were drawn to negatively charged membrane phospholipids through electrostatic interactions and extensive hydrogen bonding. LAB-4 subsequently inserted into the phosphatidyl-phosphate headgroups of the POPG and POPE bilayers via hydrophobic interactions, causing an increase in the area per lipid and a decrease in the membrane thickness. These findings demonstrated that LAB-4 exerted its antibacterial effect by inducing membrane disruption and underscored its potential as a safe and effective natural antimicrobial agent for applications in the food industry.