The Antimicrobial Peptide LJ-hep2 from Lateolabrax japonicus Exerting Activities against Multiple Pathogenic Bacteria and Immune Protection In Vivo

Abstract

Hepcidin is widely present in many kinds of fish and is an important innate immune factor. A variety of HAMP2-type hepcidins have strong antimicrobial activity and immunomodulatory functions and are expected to be developed as substitutes for antibiotics. In this study, the antimicrobial activity of Hepc2 from Japanese seabass (Lateolabrax japonicus) (designated as LJ-hep2) was investigated using its recombinant precursor protein (rLJ-hep2) expressed in Pichia pastoris and a chemically synthesized mature peptide (LJ-hep2(66–86)). The results showed that both rLJ-hep2 and synthetic LJ-hep2(66–86) displayed broad antimicrobial spectrum with potent activity against gram-negative and gram-positive bacteria, and fungi. Especially, LJ-hep2(66–86) had stronger antimicrobial activity and exhibited potent activity against several clinically isolated multidrug-resistant bacteria, including Acinetobacter baumannii, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae and Enterococcus faecium. Moreover, LJ-hep2(66–86) exerted rapid bactericidal kinetic (killed tested bacteria within 2 h), induced significant morphological changes and promoted agglutination of E. coli, P. aeruginosa and Aeromonas hydrophila. The activity of LJ-hep2(66–86) against E. coli, P. aeruginosa and A. hydrophila was stable and remained active when heated for 30 min. In addition, LJ-hep2(66–86) exhibited no cytotoxicity to the mammalian cell line HEK293T and fish cell lines (EPC and ZF4). In vivo study showed that LJ-hep2(66–86) could improve the survival rate of marine medaka (Oryzias melastigma) by about 40% under the challenge of A. hydrophila, indicating its immunoprotective function. Taken together, both rLJ-hep2 and LJ-hep2(66–86) have good prospects to be used as potential antimicrobial agents in aquaculture and medicine in the future.