Novel antimicrobial peptides derived from ribosomal protein L39 of large yellow croaker (Larimichthys crocea): promising therapeutic agents for controlling bacterial infections in aquaculture.

Abstract

Aquaculture disease management requires effective alternatives to conventional antibiotics, which contribute to antimicrobial resistance and environmental contamination through residual accumulation. Antimicrobial peptides (AMPs) are becoming the potential candidates due to their broad-spectrum activity, rapid bactericidal effects, and low resistance potential, offering sustainable solutions for aquatic disease control. To develop novel antimicrobial agents against bacterial infections in fish, this study first designed the AMP KRK12N by truncating a 12-amino acid fragment from the ribosomal protein L39 (RPL39) of large yellow croaker (Larimichthys crocea) and modifying its C-terminus with amidation. Further structural optimization through amino acid substitutions yielded derivative peptides KRK12N-1 and KRK12N-2. Structural analysis revealed that KRK12N and its derivatives primarily adopt random coil conformations in aqueous environments (PBS) but transition to α-helical structures in membrane-mimetic conditions (SDS and TFE). All peptides demonstrated broad-spectrum activity, with KRK12N-2's perfect amphipathic α-helix showing superior potency. Additionally, all three AMPs can improve the survival rate of zebrafish infected with Pseudomonas plecoglossicida. The peptides also displayed excellent biosafety, with low hemolytic activity and cytotoxicity, and maintained stable antimicrobial activity under different physicochemical conditions, such as high temperature, extreme pH, and high ionic strength. Mechanistic studies indicated that all of KRK12N, KRK12N-1, and KRK12N-2 bind lipopolysaccharide (LPS), induce membrane depolarization, and increase permeability, ultimately disrupting bacterial membrane integrity to exert their bactericidal effects. The present findings elucidate the direct bactericidal activity of the C-terminal α-helical domain of large yellow croaker RPL39, while providing a foundational basis for the development of new antimicrobial agents in aquaculture.