Study On Antibacterial Activity Of Hepcidin-2in Zebrafish Danio Rerio

Hepcidin is a cysteine-rich peptide which is structurally similar to the defensinfamily because of the four disulfide bridges in its tertiary structure. Hepcidin, alsotermed LEAP-1for liver-expressed antimicrobial peptide, was discovered by twoindependent groups while looking for antimicrobial peptides in human body fluids in2000and2001and shown to possess antimicrobial activities. Subsequent study hasuncovered that hepcidin is a long sought iron regulatory hormone involved inhereditary hemochromatosis and anemia of chronic disease, which is part of the acutephase response to infection and inflammation，thus, hepcidin plays a dual role viaacting as an iron homeostasis regulator as well as an antimicrobial agent.Two hepcidin genes present in zebrafish (GenBank accession number:AY363452and AY363453) have been identified. Zebrafish hepcidin genes display anelevated expression in the abdominal organs, skin, and heart in fish that developedsigns of infection following bacterial injection. However, antimicrobial activity ofhepcidins and their relationship between disulfide bridges and antimicrobial activityremain to be tested in zebrafish.In this report, we obtained the hepcidin-2gene from zebrafish. The ORF ofhepcidin-2is276bp long, which encodes a preprotein of92amino acids containing amature peptide of20amino acids. We then demonstrate that recombinant hepcidin-2—rHCD from zebrafish is capable of inhibiting the growth of the Gram-negativebacteria Escherichia coli and Vibrio anguillarum, and the Gram-positive bacteriaStaphylococcus aureus and Bacillus subtilis with minimum inhibitory concentrations(MICs) of18,15,13and9μM, respectively. We also show by TEM examination thatrecombinant hepcidin-2is directly cidal to the cells of E. coli and S. aureus. Moreover,we find that hepcidin-2displays affinity to LPS, LTA and PGN. All these data indicatethat hepcidin-2is both a pattern recognition molecule, capable of identifying LPS,LTA and PGN, and an antibacterial effector, capable of inhibiting the growth of bacteria. The data also show that the antibacterial activity of hepcidin-2depends uponthe disulfide bridges.