A clip domain serine proteinase plays a role in antibacterial defense but is not required for prophenoloxidase activation in shrimp

Abstract

The clip domain serine proteinases (clip-SPs) play critical roles in the signaling processes during embryonic development and in the innate immunity of invertebrates. In the present study, we identified a homolog of the clip-SP, designated as PmClipSP1, by searching the Penaeus monodon EST database (http://pmonodon.biotec.or.th), and using RACE-PCR to obtain the complete gene which contained a 1101bp open reading frame encoding 366 amino acids with a 25 amino acid signal peptide. The deduced PmClipSP1 protein sequence, which shares a predicted structural similarity to the clip-SPs of other arthropod species, appears to possess a clip domain at the N-terminus and an enzymatically active serine proteinase domain at the C-terminus. Tissue distribution analyses revealed that, at the transcript level, PmClipSP1 is mainly expressed in shrimp hemocytes, whilst temporal gene expression analyses showed that the hemocyte PmClipSP1 transcript levels were upregulated at 3h and downregulated at 6–48h following systemic Vibrio harveyi infection. RNAi-mediated silencing of the PmClipSP1 gene, by injection of double-stranded RNA (dsRNA) corresponding to the PmClipSP1 gene into shrimp, significantly reduced PmClipSP1 transcript levels, but neither significantly altered the other clip-SP and clip-SPH transcript levels nor reduced the total phenoloxidase (PO) enzyme activity in shrimp hemocytes, compared to the levels seen in the GFP dsRNA control, suggesting that PmClipSP1 is not involved in the proPO system. However, suppression of the PmClipSP1 gene led to a significant increase in the number of viable bacteria in the hemolymph (∼2.4-fold) and in the mortality rate (59%) of shrimp systemically infected with V. harveyi. These findings suggest that PmClipSP1 plays a role in the antibacterial defense mechanism of P. monodon shrimp.