Identification, characterization, and antimicrobial activity of a novel big defensin discovered in a commercial bivalve mollusc, Tegillarca granosa

Molluscs, the second largest animal phylum on earth, primarily rely on cellular and humoral immune responses to fight against pathogen infection. Although antimicrobial peptides (AMPs) such as big defensin play crucial roles in the humoral immune response, it remains largely unknown in the ecological and economic important blood clam (Tegillarca granosa). In this study, a novel big defensin gene (TgBD) was identified in T. granosa through transcripts and whole genome searching. Bioinformatic analyses were conducted to explore the molecular characteristics of TgBD, and comparisons of TgBD with those reported in other molluscs were performed by multiple alignments and phylogenetic analysis. In addition, the expression patterns of TgBD in various tissues and upon bacterial challenge were investigated while the antimicrobial activity of synthetic N-terminal domain of TgBD was confirmed in vitro by radial diffusion experiment. Results obtained showed TgBD had an open reading frame (ORF) of 369 bp, encoding a prepropeptide containing a signal peptide and a propeptide. Similar to big defensins reported in other species, TgBD consists of a hydrophobic N-terminal domain containing β1-α1-α2-β2 folds and a cysteine-rich cationic C-terminal domain with three disulfide bonds between C1-C5, C2-C4, and C3-C6. Phylogenetic analysis showed that TgBD shared 76.80% similarity to its close relative ark shell (Scapharca broughtoni). In addition, TgBD expression was observed in all tissues investigated under normal conditions and was significantly induced by injection of Vibrio parahaemolyticus. Furthermore, synthetic N-terminal peptide of TgBD exhibited strong antimicrobial activity against Gram-positive bacteria tested. Our results indicated that TgBD is a constitutive and inducible acute phase AMP, which provides a universal and prompt protection for T. granosa.