Identification and characterization of five Nk-lysins from Pseudocrossocheilus bamaensis and their diverse expression patterns in response to bacterial infection

Abstract Nk-lysin is an effector protein of cytotoxic T lymphocytes and natural killer cells. It is known to possess anti-bacterial, anti-fungal, anti-viral, and anti-tumor activity. Here we describe five Nk-lysin genes (PbNkla, PbNklb, PbNklc, PbNkld, and PbNkle) from Pseudocrossocheilus bamaensis, a rare indigenous species distributed in Guangxi, China. The open reading frames (ORFs) consisted of 426 (PbNkla), 435 (PbNklb), 369 (PbNklc), 366 (PbNkld), and 339 (PbNkle) bp nucleic acids. The surfactant-associated protein B (SapB) domain and six conserved cysteine residues were identified in each PbNkl gene. Phylogenetic analysis revealed similar results to homology comparison that PbNkla and PbNklb consist of five exons and four introns and grouped together, whereas PbNklc and PbNkld each contain four exons and three introns and formed a separate clade. PbNkle had three exons and two introns and formed an independent clade separate from the four other PbNkls. qPCR analysis demonstrated that PbNkla, PbNklc, PbNkld, and PbNkle were ubiquitously expressed in all tissues examined, whereas PbNklb was expressed only after bacterial infection. Aeromonas hydrophila challenge significantly up- and down-regulated PbNkls at different time points post-injection and in different immune-related tissues. These results suggested that PbNkls were conserved immune molecules that may be involved in the immune response to pathogen invasion.