A plant-derived antimicrobial peptide with multiple mechanisms of action exhibiting antibacterial and antibiofilm activities comparable to or superior to polymyxin B

Antimicrobial peptides (AMPs) represent promising alternatives to conventional antibiotics in the fight against multidrug-resistant pathogens. Here, we characterize the antibacterial properties and molecular mode of action of the C-terminal fragment of the <i>Medicago truncatula</i> nodule-specific cysteine-rich peptide NCR169 (NCR169C_17-38). This peptide exhibits strong bactericidal activity against a broad panel of Gram-positive and Gram-negative pathogens, including members of the ESKAPE group (<i>Enterococcus faecalis, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Escherichia coli)</i>. NCR169C_17-38 acted rapidly by permeabilizing bacterial membranes and causing severe morphological damage, outperforming polymyxin B in the rate of membrane disruption. The peptide bound selectively to anionic bacterial lipids such as cardiolipin and displayed remarkable thermal and storage stability. Moreover, NCR169C_17-38 effectively inhibited biofilm formation and eradicated pre-formed biofilms of <i>Acinetobacter baumannii</i> even at sub-MBC concentration (3.2 μM), a feature not observed for polymyxin B. Transcriptomic analysis of <i>E. coli</i> exposed to sublethal peptide doses revealed a global shutdown of bacterial metabolic and genetic functions, with 450 of 503 differentially expressed genes being downregulated. Strong repression of genes involved in translation, energy production, and cell envelope biosynthesis was accompanied by upregulation of stress- and rescue-related genes linked to membrane repair, ion transport, and oxidative stress responses. Together, these results demonstrate that NCR169C_17-38 exerts multifaceted antimicrobial effects - disrupting membranes, binding nucleic acids, and inducing transcriptional collapse - while maintaining its stability and biocompatibility. Together with its previously established antifungal activity and lack of mammalian cytotoxicity, NCR169C_17-38 represents a compelling candidate for development as a next-generation antimicrobial agent.