Characterization and functional analysis of a c-type lysozyme gene from obscure puffer Takifugu obscurus

Lysozyme (Lyz) is an alkaline enzyme that hydrolyzes mucopolysaccharides in bacteria and is highly conserved vertebrates and invertebrates. In this study, a c-type lysozyme gene (named ToLyzC) from the obscure puffer Takifugu obscurus was cloned and characterized. The full-length cDNA of ToLyzC was 432 bp, encoding 143 amino acids, with a predicted molecular mass of 16.2 kDa and a theoretical pI of 8.86. The depicted protein sequence contained a LYZ1 domain from 16 to 142 amino acids, seven conserved cysteine residues. Phylogenetic analysis indicated that ToLyzC clustered with Lyzs from other teleost fishes. Quantitative real-time PCR analysis revealed that ToLyzC mRNA was mainly expressed in the liver. The transcript level of ToLyzC gene was significantly upregulated after Staphylococcus aureus and Vibrio harveyi challenge. The optimal pH and temperature of recombinant ToLyzC protein (rToLyzC) lytic activity was detected to be 7.5 and 35 °C, respectively. rToLyzC exhibited significant antibacterial and bacterial binding activities against S. aureus, Aeromonas hydrophila, V. harveyi, and Edwardsiella tarda at different time points. In addition, the morphological changes of V. harveyi cells treated with rToLyzC were observed under scanning electron microscope, which further confirmed the antibacterial and bacteriolytic activity of rToLyzC. Taken together, our current study indicated that ToLyzC is involved in the immune response to bacterial infection in obscure puffers.