Clip domain serine proteinases and their homologs are involved in the innate immunity of invertebrates. To identify the frontline defense molecules against pathogenic infection, we isolated a novel clip domain serine proteinase (Sp-cSP) from the hemocytes of mud crab Scylla paramamosain. The full-length 1362 bp Sp-cSP contains a 1155 bp open reading frame (ORF) encoding 384 amino acids. Multiple alignment analysis showed that the putative amino acid sequence of Sp-cSP has about 52% and 51% identity with Pt-cSP2 (AFA42360) and Pt-cSP3 (AFA42361) from Portunus trituberculatus, respectively, while the similarity with other cSP sequences was lower than 30%. However, all cSP sequences possess a conserved clip domain at the N-terminal and a Tryp-SPc domain at the C-terminal. The genomic organization of Sp-cSP consists of nine exons and eight introns, with some introns containing one or more tandem repeats. RT-PCR results indicated that Sp-cSP transcripts were predominantly expressed in the subcuticular epidermis, muscle and mid-intestine, but barely detectable in the brain and heart. Further, Sp-cSP transcripts were significantly up-regulated after challenge with lipopolysaccharides (LPS), Vibrio parahaemolyticus, polyinosinic polycytidylic acid (PolyI:C) or white spot syndrome virus (WSSV). Moreover, in vitro, the recombinant Sp-cSP revealed a strong antimicrobial activity against a Gram-positive (Staphylococcus aureus) and four Gram-negative (V. parahaemolyticus, Vibrio alginolyticus, Escherichia coli, Aeromonas hydrophila) bacteria in a dose-dependent manner. Taken together, the acute-phase response to immune challenges and the antimicrobial activity assay indicate that Sp-cSP is a potent immune protector and plays an important role in host defense against pathogen invasion in S. paramamosain.
