Affiliation:
1. Guangdong Provincial Engineering Research Center for Aquatic Animal Health Assessment, Shenzhen Public Service Platform for Evaluation of Marine Economic Animal Seedings, Shenzhen Institute of Guangdong Ocean University, Shenzhen 518000, China
2. Guangdong Provincial Key Laboratory of Pathogenic Biology and Epidemiology for Aquatic Economic Animals, College of Fisheries, Guangdong Ocean University, Zhanjiang 524000, China
3. Shenzhen Base of South China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Shenzhen 518000, China
Abstract
In previous studies, natural killer lysin (NK-lysin) emerged as a crucial antimicrobial peptide (AMP) discharged by NK cells and CTLs. The sequence of NK-lysin was cloned and discovered in some fishes, but its function remains unclear. In our study, we obtained a copy of NK-lysin from the spleen of the healthy clownfish (Amphiprion ocellaris; AoNK-lysin) through cloning and proceeded to investigate its potential functions and activities. The findings showed that the AoNK-lysin gene’s open reading frame (ORF) had a length of 465 base pairs (bp) and encoded 154 amino acids (aa), which included a saposin B domain and six cysteine residues that are highly conserved, forming three intrachain disulfide bonds to carry out antimicrobial activity. The AoNK-lysin gene was widely present in different tissues, with the skin showing the highest expression, followed by the eye, intestine, and muscle. Additionally, the expression of AoNK-lysin was significantly upregulated in the immune organs (spleen, gill, intestine, and head kidney) of A. ocellaris after being challenged by Singapore group iridovirus (SGIV). Furthermore, a 399 base pair cDNA sequence that encodes the fully developed peptide of AoNK-lysin was successfully inserted into a secretion plasmid called pPIC9K. Subsequently, a significant amount of the recombinant AoNK-lysin protein was efficiently manufactured using the Pichia pastoris expression system. The antibacterial test demonstrated that the AoNK-lysin protein significantly suppressed the growth of various pathogens, particularly Streptococcus agalactiae, Streptococcus iniae, Salmonella typhi, Shigella sonnei, Pseudomonas aeruginosa, and Aeromonas caviae. The minimal inhibitory concentration (MIC) was found to be 7.81 μg/mL. Further analysis of antiviral assays showed all the viral mRNA of SGIV to be significantly reduced after AoNK-lysin protein stimuli in FHM cells. Collectively, these discoveries indicate that AoNK-lysin exhibits features of both direct pathogen-killing abilities and inhibited virus replication.
Funder
National Key Research and Development Project
National Natural Science Foundation of China
China Postdoctoral Science Foundation
Guangdong Basic and Applied Basic Research Foundation
Zhongshan Social Welfare and Basic Research Project
Shenzhen Science and Technology Program
Shenzhen Industrial Development Special Fund Project
Subject
Ecology,Aquatic Science,Ecology, Evolution, Behavior and Systematics