Cloning and immune characterization of the c-type lysozyme gene in red-spotted grouper, Epinephelus akaara

Abstract

Lysozyme is an important component of the innate immune response against pathogen infection. The gene coding for c-type lysozyme in red-spotted grouper Epinephelus akaara was cloned and designated EaClys. The complete cDNA contains a 432 bp open reading frame encoding a protein of 144 amino acids displaying 65–91% similarity with the amino acid sequences of human, mouse, chicken, and fish counterparts. Recombinant EaClys (rEaClys) was expressed in Escherichia coli, displayed antibacterial activity against Gram-positive and Gram-negative bacteria, and possessed bactericidal activity against Vibrio alginolyticus. EaClys mRNA was constitutively expressed in all tested E. akaara tissues, and its expression increased after pathogen challenge. Most notably, challenges with LPS, SGIV or V. alginolyticus upregulated EaClys mRNA expression in the head, kidney, and blood. Its expression peaked between 16 and 24 h after challenge before dropping back to the baseline level. By using recombinant cytokines as signaling pathway mimetics and blocking antibodies and chemical inhibitors as pathway inhibitors, we show that LPS-induced lysozyme release from macrophages is promoted by cytokines TNF-α and IL-1β, and dependent on NF-κB pathway activation. These data suggest that EaClys is a constitutive and inducible acute-phase protein that is involved in the innate immune defense of E. akaara, and provide new clues about the molecular mechanisms that regulate innate immune responses in fish.