Characterisation of a novel Type I crustin involved in antibacterial and antifungal responses in the red claw crayfish, Cherax quadricarinatus

Antimicrobial peptides are important immune effectors involved in mediating innate immune responses against intruding pathogens. Here, we successfully isolated and characterized a novel Type I crustin from the red claw crayfish Cherax quadricarinatus. The full-length cDNA encoded by this gene, designated CqCrs, comprised 608 bp, containing a 5′-untranslated region (UTR) of 55 bp, a 3′-UTR of 229 bp with a poly (A) tail, and an open reading frame (ORF) of 324 bp encoding a polypeptide of 107 amino acids. The deduced amino acid sequence of CqCrs exhibited a configuration typical of other crustacean Type I crustin orthologs, including one signal peptide region at the N-terminus between residues 1 and 16 and a long whey acidic protein (WAP) domain at the C-terminus between residues 60 and 107, along with a WAP-type “four-disulfide core” motif. Phylogenetic analysis showed that CqCrs was clustered first with other crustacean Type I crustins, then with other crustacean Type II crustins, and finally with other crustacean Type III crustins. Transcription of CqCrs was detected in all tissues, especially in immune tissues and was differentially induced in hemocytes post-stimulation with β-1, 3-glucan, lipopolysaccharides (LPS) and peptidoglycans (PG) at selected time-points. To clarify the biological activity of CqCrs, the recombinant CqCrs protein (rCqCrs) was constructed and expressed in Escherichia coli BL21 (DE3). Purified rCqCrs bound to diverse bacteria and inhibited the growth of different microbes to varying degrees. These findings suggest that CqCrs is involved in a specific innate immune recognition and defense mechanisms against bacterial and fungal in C. quadricarinatus.