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

## TL;DR

A plant-based peptide shows strong antibacterial and antibiofilm effects, working better than some existing antibiotics and targeting bacteria in multiple ways.

## Contribution

A new plant-derived antimicrobial peptide with multiple mechanisms of action and superior antibiofilm activity compared to polymyxin B.

## Key findings

- The peptide shows potent antibacterial activity against eight pathogens in various growth media.
- It effectively inhibits and eradicates biofilms, outperforming polymyxin B at sub-MBC concentrations.
- Transcriptomic analysis reveals it disrupts bacterial metabolism and gene expression.

## Abstract

•Potent antibacterial activity against eight pathogens across different growth media.•Strong biofilm inhibition and bactericidal efficacy on biofilm-embedded cells.•Biofilm eradication at sub-MBC outperforming polymyxin B.•Lipid and nucleic acid binding, suggesting multiple intracellular modes of action.•Global transcriptomic changes including membrane, stress, and metabolism genes.

Potent antibacterial activity against eight pathogens across different growth media.

Strong biofilm inhibition and bactericidal efficacy on biofilm-embedded cells.

Biofilm eradication at sub-MBC outperforming polymyxin B.

Lipid and nucleic acid binding, suggesting multiple intracellular modes of action.

Global transcriptomic changes including membrane, stress, and metabolism genes.

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 Medicago truncatula nodule-specific cysteine-rich peptide NCR169 (NCR169C17–38). This peptide exhibits strong bactericidal activity against a broad panel of Gram-positive and Gram-negative pathogens, including members of the ESKAPE group (Enterococcus faecalis, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Escherichia coli). NCR169C17–38 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, NCR169C17–38 effectively inhibited biofilm formation and eradicated pre-formed biofilms of Acinetobacter baumannii even at sub-MBC concentration (3.2 μM), a feature not observed for polymyxin B. Transcriptomic analysis of E. coli 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 NCR169C17–38 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, NCR169C17–38 represents a compelling candidate for development as a next-generation antimicrobial agent.

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