Mudskipper (Boleophthalmus pectinirostris) Hepcidin-1 and Hepcidin-2 Present Different Gene Expression Profile and Antibacterial Activity and Possess Distinct Protective Effect against Edwardsiella tarda Infection

Hepcidins are small cysteine-rich antimicrobial peptides that play an important role in fish immunity against pathogens. Most fish species have two or more hepcidin homologs that have distinct functions. This study investigated the immune functions of mudskipper (Boleophthalmus pectinirostris) hepcidin-1 (BpHep-1) and hepcidin-2 (BpHep-2) in vitro and in vivo. Upon infection with Edwardsiella tarda, the expression of BpHep-1 and BpHep-2 mRNA in immune tissues was significantly upregulated, but the expression profiles were different. Chemically synthesized BpHep-1 and BpHep-2 mature peptides exhibited selective antibacterial activity against various bacterial species, and BpHep-2 exhibited a stronger antibacterial activity and broader spectrum than BpHep-1. BpHep-1 and BpHep-2 both inhibited the growth of E. tarda in vitro, with the latter being more effective than the former. In addition, both peptides induced hydrolysis of purified bacterial genomic DNA (gDNA) or gDNA in live bacteria. In vivo, an intraperitoneal injection of 1.0 mu g/g BpHep-2 significantly improved the survival rate of mudskippers against E. tarda infection compared with 0.1 mu g/g BpHep-2 or 0.1 and 1.0 mu g/g BpHep-1. Similarly, only BpHep-2 treatment effectively reduced the tissue bacterial load in E. tarda-infected mudskippers. Furthermore, treatment with 1.0 or 10.0 mu g/ml BpHep-2 promoted the phagocytic and bactericidal activities of mudskipper monocytes/macrophages (MO/M phi). However, only the highest dose (10.0 mu g/ml) of BpHep-1 enhanced phagocytosis, and BpHep-1 exerted no obvious effects on bactericidal activity. In conclusion, BpHep-2 is a stronger bactericide than BpHep-1 in mudskippers, and acts not only by directly killing bacteria but also through an immunomodulatory function on MO/M phi.