Dual Function Characterisation of the 4-Cysteine Hepcidin in the Antarctic Toothfish Dissostichus mawsoni

Hepcidin is a cysteine-rich peptide that functions both as an antimicrobial peptide and a central regulator of iron homeostasis in vertebrates. Structurally, it consists of an N-terminal signal peptide, a propeptide region and a C-terminal bioactive mature peptide. The canonical mature hepcidin contains eight conserved cysteine residues that form four disulfide bonds, stabilising a characteristic β-sheet hairpin-like structure. In the Antarctic toothfish (Dissostichus mawsoni), which inhabits extremely cold marine environments, a unique hepcidin variant was identified possessing only four cysteine residues, designated dm_4cyshep. To evaluate the antimicrobial function of the dm_4cyshep peptide, the minimum inhibitory concentration (MIC) assays and flow cytometry analyses were performed. The peptide demonstrated binding affinity and bactericidal activity against a range of Gram-positive and Gram-negative bacteria, with distinct MIC values observed for different strains. Furthermore, overexpression of dm_4cyshep in a hepcidin-deficient zebrafish model markedly reduced systemic iron accumulation, indicating a conserved role in iron regulation. These results demonstrate that despite the reduced number of cysteine residues, dm_4cyshep retains both antimicrobial and iron-modulatory functions, underscoring its potential as an immune effector molecule. This study enhances the understanding of the structural and functional diversity of fish hepcidins and identifies a promising candidate for improving disease resistance in aquaculture.