A new crustin homologue (SpCrus6) involved in the antimicrobial and antiviral innate immunity in mud crab, Scylla paramamosain.

Crustins play important roles in defending against bacteria in the innate immunity system of crustaceans. In present study, we identified a crustin gene in Scylla paramamosain, which was named as SpCrus6. The ORF of SpCrus6 possessed a signal peptide sequence (SPS) at the N-terminus and a WAP domain at the C-terminus. And there were 5 Proline residues, 5 Glycine and 4 Cysteine residues between SPS and WAP domain in SpCrus6. These features indicated that SpCrus6 was a new member of crustin family. The SpCrus6 mRNA transcripts were up-regulated obviously after bacteria or virus challenge. These changes showed that SpCrus6 was involved in the antimicrobial and antiviral responses of Scylla paramamosain. Recombinant SpCrus6 (rSpCrus6) showed strong inhibitory abilities against Gram-positive bacteria (Bacillus megaterium, Staphylococcus aureus, and Bacillus subtilis). But the inhibitory abilities against four Gram-negative bacteria (Vibrio parahemolyticus, Vibrio alginolyticus, Vibrio harveyi and Escherichia coli) and two fungi (Pichia pastoris and Candida albicans) were not strong enough. Besides, rSpCrus6 could strongly bind to two Gram-positive bacteria (B. subtilis and B. megaterium) and three Gram-negative bacteria (V. alginolyticus, V. parahemolyticus, and V. harveyi). And the binding levels to S. aureus and two fungi (P. pastoris and C. albicans) were weak. The polysaccharides binding assays' results showed rSpCrus6 had superior binding activities to LPS, LTA, PGN and β-glucan. Through agglutinating assays, we found rSpCrus6 could agglutinate well three Gram-positive bacteria (S. aureus, B. subtilis and B. megaterium). And the agglutinating activities to Gram-negative bacteria and fungi were not found. In the aspect of antiviral functions, rSpCrus6 could bind specifically to the recombinant envelop protein 26 (rVP26) of white spot syndrome virus (WSSV) but not to recombinant envelop protein 28 (rVP28), whereas GST protein could not bind to rVP26 or rVP28. Besides, rSpCrus6 could suppress WSSV reproduction to some extent. Taken together, SpCrus6 was a multifunctional immunity effector in the innate immunity defending response of S. paramamosain.