Bacteria-Induced Oligopeptide Assembly Enables Effective Bacterial Killing with Low Cytotoxicity and Hemolysis

Bacteria-induced on-membrane assembly of oligopeptides showed the potential for balancing their persistent conflict between antibacterial activity and biocompatibility. However, molecular determinants linking the peptide sequence, assembly behavior, antibacterial efficacy, and biocompatibility remain insufficiently understood. Here, we investigated a series of heptapeptides, (RX)₃R (X = A, I, F), and demonstrated the significant influence of hydrophobic residues on bacteria-induced assembly, antibacterial performance, cytotoxicity, and hemolytic activity. Negatively charged liposomes or bacterial membranes could induce the assembly of (RI)₃R and (RF)₃R into nanofibers, which in turn exhibited enhanced antibacterial efficacy against Gram-negative <i><i>Escherichia coli</i></i> with minimum inhibitory concentrations (MICs) of 60 μM but poor antibacterial activity on Gram-positive <i>Staphylococcus aureus</i> (MICs: 400-500 μM). Importantly, both peptides demonstrated excellent biocompatibility, with cytotoxicity thresholds of 600 μM (RI)₃R and 2000 μM (RF)₃R and hemolysis thresholds up to 2000 and 4000 μM, respectively. These findings provide valuable insights for the rational design of antibacterial oligopeptides.