Hepcidin was first identified as an antimicrobial peptide present in human serum 
and urine. It was later demonstrated that hepcidin is the long sought hormone 
that regulates iron homeostasis in mammals. The native peptide of 25 amino acids 
(Hepc25) contains four disulfide bridges that maintain a β-hairpin motif. The 
aim of the present study was to assess whether the intramolecular disulfide 
bridges are necessary for Hepc25 antimicrobial activity. We show that a 
synthetic peptide corresponding to human Hepc25, and which contains the four 
disulfide bridges, has an antibacterial activity against several strains of 
Gram-positive and Gram-negative bacteria. On the contrary, a synthetic peptide 
where all cysteines were replaced by alanines (Hepc25-Ala) had no detectable 
activity against the same strains of bacteria. In a further step, the mode of 
action of Hepc25 on Escherichia coli was studied. SYTOX Green uptake was used to 
assess bacterial membrane integrity. No permeabilization of the membrane was 
observed with Hepc25, indicating that this peptide does not kill bacteria by 
destroying their membranes. Gel retardation assay showed that the Hepc25 binds 
to DNA with high efficiency, and that this binding ability is dependent on the 
presence of the intramolecular disulfide bridges. Reduction of Hepc25 or 
replacement of the eight cysteines by alanine residues led to peptides that were 
no longer able to bind DNA in the in vitro assay. Altogether, these results 
demonstrate that Hepc25 should adopt a three-dimensional structure stabilized by 
the intramolecular disulfide bridges in order to have antibacterial activity.