Bactericidal and Antibiofilm Activities of HT-2-1-3, a Vespidae Venom-Derived Antimicrobial Peptide, Against Streptococcus mutans UA159

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Background: Dental caries is a biofilm-associated disease in which Streptococcus mutans plays a central role. This study evaluated HT-2-1-3, a Vespidae venom-derived antimicrobial peptide, as a potential topical anti-caries lead. Methods: Candidate peptides were synthesized by Fmoc solid-phase peptide synthesis and characterized by RP-HPLC and ESI-MS. Antibacterial activity against S. mutans UA159 was assessed using MIC, MBC, growth-curve, and time-kill assays. Membrane damage, antibiofilm activity, cytocompatibility, hemolysis, and preliminary oral safety in mice were further evaluated. Results: HT-2-1-3 showed the strongest activity among the tested peptides, with MIC and MBC values of 2 µg/mL. It inhibited bacterial growth in a concentration-dependent manner and achieved complete killing at 4×MIC (8 µg/mL) within 2 h. Mechanistic assays showed rapid membrane depolarization, increased PI uptake, and concentration-dependent release of extracellular DNA and proteins. MD simulations further indicated that HT-2-1-3 entered the model S. mutans UA159 membrane at 1857 ns, supporting membrane permeabilization as a central component of its bactericidal activity. Scanning electron microscopy revealed dose-dependent membrane disruption, cell collapse, and rupture. HT-2-1-3 inhibited biofilm formation by approximately 58–63% and eradicated up to 89.0% of mature biofilms. Hemolysis remained below 4% at concentrations up to 64×MIC (128 µg/mL), and mammalian-cell viability exceeded 95% at concentrations up to 16×MIC (32 µg/mL). Repeated oral administration caused no obvious gingival irritation in mice. Conclusions: HT-2-1-3 showed strong bactericidal and antibiofilm activity against S. mutans UA159 and favorable preliminary biocompatibility. Its mode of action may involve membrane disruption, and it showed favorable preliminary biosafety. These findings support further optimization of HT-2-1-3 as a topical antimicrobial candidate for caries prevention and control.