Dual-Target Antimicrobial Strategy Combining Cell-Penetrating Protamine Peptides and Membrane-Active ε-Poly-L-lysine

Dental caries is a major global health issue associated with biofilm formation by Streptococcus mutans (S. mutans). Conventional antimicrobials often fail to eliminate biofilms due to their structural resistance, highlighting the need for new strategies. This study investigated the antibacterial and antibiofilm effects of protamine peptides (PPs), which are cell-penetrating antimicrobial peptides derived from salmon protamine, alone and in combination with antimicrobial agents. Antimicrobial susceptibility was evaluated using alamarBlue® and colony count assays, while biofilm formation was analyzed using crystal violet staining, confocal microscopy, and extracellular polysaccharide (EPS) quantification. PP exhibited moderate antibacterial activity but strongly suppressed EPS accumulation and biofilm development, leading to a flattened biofilm structure. Cotreatment with ε-poly-L-lysine (PL) significantly enhanced antibacterial and antibiofilm effects compared with either agent alone, whereas this effect was not observed with other cationic polymers. Fluorescence imaging revealed that PL promoted the intracellular localization of PP without increasing membrane damage, indicating a cooperative mechanism by which PL enhances membrane permeability and PP targets intracellular sites. These findings demonstrate that combining a cell-penetrating peptide with a membrane-active agent is a novel approach to overcome bacterial tolerance. The PP–PL combination effectively suppressed S. mutans growth and biofilm formation through dual action on membranes and EPS metabolism, offering a promising basis for the development of peptide-based preventive agents and biofilm-resistant dental materials.