Involvement of a newly identified atypical type II crustin (SpCrus5) in the antibacterial immunity of mud crab Scylla paramamosain.

Crustins, the main AMP family in Crustacea, are generated as isoforms in many species and implicated in innate immune responses, but their detailed molecular mechanisms on susceptible bacteria remain largely unclear. Type II and type I crustins are distinguished by glycine-rich region (GRR), which is a major marker motif, and some type II crustins exhibit stronger antibacterial activities than their GRR deletion mutants. In the present study, a novel crustin, namely, SpCrus5 , was functionally characterized from a commercially valuable crab Scylla paramamosain . Sp Crus5 contained a typical cysteine-rich domain at the N-terminus, a conserved WAP domain in the center, and a special GRR at the C-terminus, which is located in a site that differs from that of GRRs in typical type II crustins found between signal peptides and cysteine-rich domains. Sp Crus5 shared high similarities with most type II crustins, and it was more closely related to type II crustins than to other retrieved crustins. SpCrus5 was predominantly expressed in gills and remarkably upregulated after the crabs were challenged with Vibrio parahemolyticus or Staphylococcus aureus , suggesting that SpCrus5 might participate in antibacterial immune responses. To further elucidate how this C-terminal GRR affects the function of Sp Crus5, we harvested a GRR deletion mutant ( Sp Crus5-ΔGRR) by deleting the GRR. Liquid growth inhibition assays demonstrated that the antimicrobial activity of Sp Crus5 was stronger than that of Sp Crus5-ΔGRR, and the antibacterial spectrum of the former toward Gram-negative bacteria was broader than that of the latter. Binding assays revealed that the microorganism-binding ability and polysaccharide-binding activity of Sp Crus5 were stronger than those of Sp Crus5-ΔGRR. Sp Crus5 or Sp Crus5-ΔGRR agglutinated all tested Gram-positive bacteria. Therefore, the antibacterial activities of Sp Crus5 were stronger and broader than those of Sp Crus5-ΔGRR, and the binding ability and agglutination activity might contribute to the antimicrobial activity of Sp Crus5. These results revealed that the C-terminal GRR was necessary to produce an efficient antibacterial activity of Sp Crus5. Sp Crus5 was highly identical with most type II crustins and it functioned as many type II crustins did, indicating that Sp Crus5 was more likely an atypical type II crustin than a type I crustin. This study revealed that Sp Crus5 participated as an essential antimicrobial effector in immune responses and provided new insights into the underlying mechanisms of the sequence and function diversity of crustins. [ABSTRACT FROM AUTHOR]