Isolation, gene cloning and expression profile of a pathogen recognition protein: A serine proteinase homolog (Sp-SPH) involved in the antibacterial response in the crab Scylla paramamosain

Feb 16, 2010Developmental and comparative immunology

Isolation, gene cloning, and activity of a protein involved in antibacterial defense in the crab Scylla paramamosain

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Abstract

A full-length cDNA sequence of 1298 bp encoding a serine proteinase homolog (Sp-SPH) was identified in the crab Scylla paramamosain.

Sp-SPH shares 83.8% identity with similar proteins from other crab species.
The Sp-SPH transcripts are present in various tissues including the eye stalk, gill, and brain.
High expression of Sp-SPH occurs at different developmental stages such as embryo and juvenile.
Prophenoloxidase is found in embryos, zoea, juvenile, and adult crabs, but less in megalopa.
Sp-SPH mRNA expression significantly increases in hemocyte and epidermis at 24 hours and in gill at 96 hours after bacterial challenge.

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To identify the frontline defense molecules against microbial infection in the crab Scylla paramamosain, a live crab pathogenic microbe, Vibrio parahaemolyticus, was recruited as an affinity matrix to isolate innate immune factors from crab hemocytes lysate. Interestingly, a serine proteinase homolog (Sp-SPH) was obtained together with an antimicrobial peptide-antilipopolysaccharide factor (Sp-ALF). We then determined the full-length cDNA sequence of Sp-SPH, which contained 1298bp with an open reading frame of 1107bp encoding 369 amino acid residues. Multiple alignment analysis showed that the deduced amino acid sequences of Sp-SPH shared overall identity (83.8%) with those of SPH-containing proteins from other crab species. Tissue distribution analysis indicated that the Sp-SPH transcripts were present in various tissues including eye stalk, subcuticular epidermis, gill, hemocyte, stomach, thorax ganglion, brain and muscle of S. paramamosain. The Sp-SPH was highly expressed in selected different development stages including embryo (I, II, III and V), zoea (I), megalopa, and juvenile. Importantly, the prophenoloxidase was also present in the embryos, zoea, juvenile and adult crabs, but relatively lower in megalopa compared to those of other stages. Furthermore, the Sp-SPH mRNA expression showed a statistically significant increase (P<0.05) in both hemocyte and subcuticular epidermis at 24h, and in gill at 96h after challenge of V. parahaemolyticus determined by quantitative real-time PCR. Taken together, the live-bacterial-binding activity and the acute-phase response against bacterial infection of Sp-SPH suggested that it might function as an innate immune recognition molecule and play a key role in host defense against microbe invasion in the crab S. paramamosain.

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Isolation, gene cloning and expression profile of a pathogen recognition protein: A serine proteinase homolog (Sp-SPH) involved in the antibacterial response in the crab Scylla paramamosain

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