Cloning, subcellular localization and antibacterial functional analysis of NK-lysin in yellow drum (Nibea albiflora)

Vibrio harveyi is the primary pathogenic bacteria affecting Nibea albiflora aquaculture. In a previous phase, our laboratory intentionally exposed N. albiflora to V. harveyi and analyzed the outcomes using a combination of genome-wide association study (GWAS) and RNA-seq. The results revealed that the antimicrobial peptide NK-lysin (YdNkl-1) was a candidate gene for resistance to V. harveyi disease in N. albiflora. To investigate the role of the antimicrobial peptide NK-lysin in N. albiflora's antimicrobial immunity, we screened the YdNkl-1 gene from the transcriptome database. The full-length cDNA of YdNkl-1 gene is 508 bp, with an open reading frame (ORF) of 477 bp, encoding 158 amino acids. The deduced amino acid sequence of YdNkl-1 contains a signal peptide (1st-22nd amino acids) and a Saposin B domain (50th-124th amino acids), akin to mammalian NK-lysin. Phylogenetic tree analysis confirmed that the NK-lysin of teleost fish clustered into a single species, and YdNkl-1 was most closely related to Larimichthys crocea. Subcellular localization showed that YdNkl-1 was distributed in cytoplasm and nucleus of yellow drum kidney cells. Furthermore, YdNkl-1 mRNA transcripts were significantly up-regulated in the skin, gill, intestine, head-kidney, liver, and spleen after V. harveyi infection, suggesting a critical role in N. albiflora's defense against V. harveyi infection. Additionally, we purified and observed the YdNkl-1 protein, which exhibited a potent membrane-disrupting effect on V. harveyi, Pseudomonas plecoglossicida, Vibrio parahaemolyticus, Escherichia coli and Bacillus subtilis. These findings underscore the significance of NK-lysin in N. albiflora's resistance to V. harveyi infection and provide new insights into the crucial role of NK-lysin in the innate immunity of teleost fishes.