Identification, distribution, bactericidal and immunoregulatory activities of NK-lysins in grass carp (Ctenopharyngodon idella)

NK-lysins, which are antimicrobial peptide mainly produced by cytotoxic T lymphocytes and natural killer cells, play an important role in immune defense against infection. In this study, using CiNkla gene as template, 2 highly homologs CiNklb and CiNklc were further identified in grass carp. The three NK-lysin transcripts were ubiquitously distributed in various tissues, and CiNkla and CiNklb have the highest transcripts in spleen while CiNklc has the highest transcript in gill. Upon challenge with Aeromonas hydrophila, the transcripts of CiNkla and CiNklb were significantly increased in intestine, whereas transcript of CiNklc increased in liver. The concentration of steady-state CiNkla protein was highest in spleen in healthy fish, while upon challenge with A. hydrophila, highest level of CiNkla protein was detected in head kidney at 6 h post infection (hpi), and in liver at either 24 hpi or 48 hpi. The recombinant CiNkla and CiNklb purified from Pichia pastoris show direct bactericidal activity against both Gram-negative and Gram-positive bacterial pathogens. The A. hydrophila treated with CiNkla showed morphological change, release of intracellular contents and genome DNA degradation. Supplementation of CiNkla or CiNklb to cultured head kidney derived macrophages (HKDMs) of grass carp changes the transcription of inflammatory factors and chemokines. In vivo stimulation of fish with recombinant CiNkla or CiNklb confirmed both molecules could increase transcription of chemokines and cytokines in tissues, and liver was sensitive to stimulation and induced into an inflammation condition at earlier timepoints. Our results indicate that transcription and expression of NK-lysins are modulated by bacterial infection, and CiNkla and CiNklb could modulate host immune defense.