Hepcidin gene of Indian major carp, Labeo rohita: Molecular, structural and functional characterization, and antibacterial activity of recombinant hepcidin

Abstract

Hepcidin, an antimicrobial peptide produced in the liver, also regulates iron balance and recycling in vertebrates. To study the antibacterial activity of Labeo rohita hepcidin (LrHep), full-length cDNA of LrHep was cloned. The 800 bp amplicon contained an open reading frame of 276 bp, encoding 91 amino acids including a signal peptide of 24 amino acid residues. The 3D structure and domain architecture of LrHep were predicted. Phylogenetic analysis of the LrHep sequence showed that it formed a sister group with another carp, Sinocyclocheilus rhinocerous. LrHep has eight cysteine residues at C73, C76, C77, C79, C80, C85, C88 and C89 positions conserved throughout evolution. Besides cysteine residues, like in all teleosts, A24, P26, E34 and R63 were also found to be conserved in LrHep. Expression analysis of LrHep using 12 different tissues of naïve rohu juveniles revealed that its expression is being the highest in the liver followed by anterior kidney. To study the role of LrHep during disease, its expression kinetics was observed in three infection models (Aeromonas hydrophila, poly I:C, and Argulus siamensis, representatives of bacterial, viral, and parasitic infections, respectively). Significant up-regulation of LrHep was observed in the liver in all the three experimental groups (at 12 h with A. hydrophila infection, 3 h with poly I:C stimulation and 24 h with A. siamensis infection). Significant up-regulation in anterior kidney was observed at 3 and 6 h only in A. hydrophila infected fish. The antimicrobial activity of LrHep was evaluated both in vitro and in vivo by producing recombinant hepcidin (approximately 11 kDa) of rohu (rLrHep) in Escherichia coli. The recombinant LrHep inhibited growth of the bacteria, Edwardsiella tarda and A. hydrophila, in vitro at concentrations of 50 and 100 μg (4.54 and 9.09 μM), respectively, at 6 h post-incubation. Administration of rLrHep to rohu juveniles rendered protection against A. hydrophila challenge with a relative percent survival (RPS) of 73%. Overall, the findings of this study suggest that owing to its potential antimicrobial activity, LrHep could be an effective immunomodulator providing protection against A. hydrophila infection.