Molecular characterization and antibacterial activity of a novel i-type lysozyme from Amphioctopus fangsiao

As a bacterial terminator, lysozyme is one of the most powerful weapons for cephalopoda in protecting its soft body from pathogen invasion. In this study, a novel i-type lysozyme homology was identified from Amphioctopus fangsiao (AF-LYZ2). The cDNA of AF-LYZ2 contained 736 bp with a ORF of 444 bp encoding a polypeptide of 143 amino acids. The polypeptide chain had a signal peptide of 1(M)-24(A) and a typical destabilase domain located from 32(P) to 139(C). Multiple sequence alignments showed that the amino acid sequence of AF-LYZ2 contained the specific motif "CL(E/L/R/H/Q)C(I/M/L)C″ and two catalytic residues Glu43 and Asp55 highly conserved in i-type lysozymes, indicating that it might be a new member of the i-type lysozyme subfamily. The predicted 3D structure of AF-LYZ2 showed that it contained seven α-helices, random coiled-coils and six disulfide bonds. Tissue distribution analysis revealed that AF-LYZ2 could be expressed in up to nine tissues, where it was highly expressed in skin, white body and hemocyte. After A. fangsiao was challenged by Vibrio anguillarum, the mRNA levels of AF-LYZ2 increased significantly in these three tissues within 24 h. The recombinant protein rAF-LYZ2 expressed in E. coli BL21(DE3) had the antimicrobial activities against Gram-positive higher than against Gram-negative bacteria, probably which was affected by pH and temperature. Further finding was the thermal stability of rAF-LYZ2, working in an enzymatic independent way. The damage mechanism of rAF-LYZ2 was analyzed by PI staining and SEM, and showed that rAF-LYZ2 could enhence membrane permeability and promote the degradation of bacterial cell wall integrity. Taken together, these results suggested that AF-LYZ2 played an important role in the process of A. fangsiao innate immune defense, and provided new light to understand the antimicrobial mechanism of lysozyme from cephalopoda.