Barbel steed (Hemibarbus labeo) NK-lysin protects against Aeromonas hydrophila infection via immunomodulatory activity.

NK-lysins (NKLs) are a family of multifunctional antimicrobial peptides that have activity against various microorganisms. However, the immunomodulatory activity of NKL in fish remains unclear. In this study, the cDNA sequence of barbel steed (Hemibarbus labeo) NKL gene was cloned. Barbel steed NKL amino acid sequence comprised a signal peptide and a mature peptide. The saposin B domain in the mature peptide has six conserved cysteines that form three disulfide bonds. Phylogenetic analysis showed that the barbel steed NKL was most closely related to that of the common carp (Cyprinus carpio) NKL. Differential expression analysis showed that the barbel steed NKL gene was expressed in all tested tissues, with the highest expression in the spleen. In response to Aeromonas hydrophila infection, NKL was significantly upregulated in the liver, spleen, head kidney, and gill. The barbel steed NKL showed strong antibacterial activity against Vibrio parahaemolyticus, V. alginolyticus, V. vulnificus, and Listeria monocytogenes. However, NKL had no antibacterial activity against the pathogenic bacteria A. hydrophila. Lactate dehydrogenase release assays showed that NKL damaged the V. parahaemolyticus cell membrane. NKL significantly increased barbel steed survival rate after A. hydrophila infection and upregulated IL-1β and TNF-α expression in the spleen and head kidney. NKL induced monocyte/macrophage chemotaxis and enhanced the respiratory burst and proinflammatory cytokine expression. Our study shows that fish NKL exhibits immunomodulatory effects and protects the host from pathogenic infections independent of direct bacterial clearance.