Characterization of a type-I crustin with broad-spectrum antimicrobial activity from red swamp crayfish Procambarus clarkii

Crustins are a family of antimicrobial peptides mainly identified in crustaceans and characterized by a whey acidic protein (WAP) domain and an additional glycine-, cysteine-, or proline-rich region. In this study, we identified and characterized PcCru, a new crustin isolated from red swamp crayfish Procambarus clarkii. The open reading frame of PcCru was 333 base pairs long and encoded a 110-residue polypeptide, which contained a signal peptide, a cysteine-rich region, and a WAP domain. The architecture and phylogenetic analysis suggested that PcCru was a new member of the type-I crustin family. PcCru was highly expressed in hemocytes and was significantly induced by viral and bacterial stimulations at both the translational and transcriptional levels. The titer of PcCru in circulating plasma was also increased considerably by bacterial challenge. Recombinant PcCru from both prokaryotic and eukaryotic expression systems were generated, and the proteins exhibited broad-spectrum antimicrobial activity. Furthermore, PcCru protected crayfish from infection by pathogenic bacteria Aeromonas hydrophila in vivo. This study provided new information emphasizing the important role of the crustin family in the crustacean antibacterial immune response.