Characterization and functional analysis of prophenoloxidase system-associated genes from the black tiger shrimp penaeus monodon / Walaiporn Charoensapsri

Abstract

The prophenoloxidase (proPO) activating system plays an essential role in the immune defence against microbial infections in many invertebrates. In the present study, two PPAE genes (designated PmPPAE1 and PmPPAE2) and a novel proPO (PmproPO2) gene were identified from hemocytes of the black tiger shrimp, Penaeus monodon. Sequence analysis revealed that PmPPAE1 exhibited the highest amino acid sequence similarity of 70% to a PPAE of the freshwater crayfish Pacifastacus leniusculus, whilst PmPPAE2 showed 51% amino acid sequence similarity to the insect Manduca sexta PAP1. Analysis of the PmproPO2 sequences showed that PmproPO2 shared a 67% sequence identity to the PmproPO1. Tissue expression analysis revealed that PmPPAE1, PmPPAE2, PmproPO1 and PmproPO2 mRNA transcripts were mainly expressed in hemocytes. Analysis of their larval developmental gene expression revealed that PmPPAE1 and PmproPO2 transcripts were expressed in all larval stages (nauplius, protozoea, mysis and post-larvae), whereas PmPPAE2 and PmproPO1 transcripts were mainly expressed in the late stages of larval development (mysis and post-larvae). Double-stranded RNA(dsRNA)-mediated gene suppression of PmPPAE1and PmPPAE2 resulted in a significant reduction in the respective transcript levels and resulted in a significant decreased in total PO activity in the PmPPAE1 (37%) and PmPPAE2 (41%) knockdown shrimps as compared with saline injected group. Experimental infection of PmPPAE knockdown shrimps with the highly pathogenic bacterium Vibrio harveyi significantly increased the cumulative mortality and the number of bacterial colonies in the silenced shrimps. These results indicate that PmPPAE1 and PmPPAE2 participate in the proPO system and also play an important role in the shrimp immune defence against V. harveyi infection. Gene silencing of PmproPO1 and PmproPO2 by RNA interference (RNAi) resulted in a significant decrease in the respective endogenous proPO mRNA levels in hemocytes and a reduction of total PO activity by 75 and 73%, respectively. Experimental infection of P. monodon with the V. harveyi revealed that the PmproPO silenced shrimps were more susceptible to bacterial infection than the control GFP dsRNA and saline injected shrimps, suggesting that the two proPOs are important components in the shrimp immune system and play the crucial role in the defence against V. harveyi infection. In addition, the mature protein of PmPPAE1 was expressed in Escherichia coli and insect cell expression system, but the obtained recombinant proteins were not stable. The SP domain of PmPPAE1 was expressed in E. coli system. The recombinant SP domain of PmPPAE1 was used as an immunogen to generate the antibody against SP domain of PmPPAE1 and western blot analysis demonstrated that the endogenous PmPPAE1 was found only in hemocytes but not in cell-free plasma of P. monodon.