In silico design and In Vitro validation of WK15: a promising antimicrobial peptide for aquaculture applications

Antimicrobial peptides serve as an innate defence mechanism against various pathogens. They are being explored as potential alternatives to antibiotics and could play an important role in addressing antimicrobial resistance in aquaculture. In this study, a novel 15-mer antimicrobial peptide, WK15, was rationally designed from the alpha-subunit of rainbow trout haemoglobin. The peptide is amphipathic, exhibits strong membrane interactions, and has a net charge of + 5. Molecular docking showed that WK15 binds strongly to aerolysin, a virulence protein of Aeromonas sobria. Its antimicrobial activity was tested against a range of bacterial pathogens, including Staphylococcus epidermidis, Lactococcus garvieae, Staphylococcus aureus, Edwardsiella tarda, Aeromonas sobria, Yersinia ruckeri, and Aeromonas hydrophila. The peptide exhibited broad-spectrum antimicrobial activity, with minimum inhibitory concentration values ranging from 22.6 µM to 500 µM and minimum bactericidal concentration values from 45.2 µM to 700 µM. Additionally, WK15 remained stable and effective at higher temperatures and under elevated salt and serum levels. DNA-binding assays confirmed its concentration-dependent interaction with bacterial DNA in vitro, suggesting that DNA binding may represent a potential secondary mechanism. The peptide demonstrated negligible cytotoxicity toward the Epithelioma Papulosum Cyprini cell line and low haemolytic activity against fish erythrocytes, indicating favourable biocompatibility and reduced host-cell toxicity. Scanning electron microscopy analysis of S. aureus treated with WK15 revealed significant morphological alterations consistent with membrane-targeting activity. Collectively, these findings highlight that WK15 is a promising candidate with high stability and negligible host toxicity, underscoring its potential as an alternative to conventional antibiotics in aquaculture.