Mode of antimicrobial action of plantaricin EvF and the design of activity-enhanced mutants

Two-peptide bacteriocins are ribosomally-synthesized antimicrobial peptides with promising potential in food preservation. However, their structure-activity relationships and strategies for rational enhancement remain insufficiently understood. To address this, this study investigates the antimicrobial mode of action of plantaricin EvF and designs activity-enhanced mutants. Membrane permeability assays, cell viability assessments, and membrane depolarization assays showed that plantaricin EvF disrupts bacterial membrane integrity by accumulating at higher concentrations, resulting in the loss of membrane potential and leakage of cellular contents, ultimately leading to bacterial death. Liposome leakage experiments, simulating bacterial membranes, further confirmed that plantaricin EvF directly targets the membranes. Atomic force microscopy revealed that plantaricin EvF, at 0.5 × MIC concentration, forms dense nanometer-sized pores (10–30 nm) in the membrane, causing irregular structural damage. Mutant design studies identified a strategy to enhance the antimicrobial activity of plantaricin EvF, resulting in the development of the activity-enhanced mutant, plantaricin EsF. The mutant exhibited a MIC of 2 μmol/L, which is a 75 % reduction compared to plantaricin EvF, along with superior pH adaptability, thermal stability, and excellent bactericidal kinetics. These findings provide valuable insights into the optimization of plantaricin EvF and its mutants for the development of more effective antimicrobial peptides.