The
prophenoloxidase (proPO) activating system is an important innate immune response against microbial
infections in invertebrates. The major
enzyme,
phenoloxidase (PO), is synthesized as an inactive precursor and its activation to an active
enzyme is mediated by a cascade of
clip domain
serine proteinases (
clip-SPs). In this study, a
cDNA encoding a proPO activating
enzyme (PPAE) from the black tiger shrimp, Penaeus monodon, designated as PmPPAE1, was cloned and characterized. The full-length
cDNA contains an open reading frame (ORF) of 1392bp encoding a predicted
protein of 463
amino acids including an 18
amino acid signal peptide. The PmPPAE1
protein exhibits a characteristic sequence structure of
clip-SPs consisting of the
clip domain at the N-terminus and a SP domain at the C-terminus. Sequence analysis showed that PmPPAE1 exhibited the highest amino acid sequence similarity (70%) to a PPAE of the crayfish, Pacifastacus leniusculus. PmPPAE1
mRNA is abundantly expressed in hemocytes, and this is regulated after systemic Vibrio harveyi
infection supporting that it is an immune-responsive gene. RNA interference-mediated suppression of PmPPAE1, performed by injection of
double-stranded RNA (dsRNA) corresponding to the PmPPAE1 gene into shrimp, resulted in a significant reduction of PmPPAE1 but not other
clip-SP and related gene transcript levels of P. monodon, suggesting gene-specific knockdown. RNAi-mediated silencing of PmPPAE1 gene significantly decreased the total PO activity (36.7%) in shrimp and additionally increased the mortality of V. harveyi infected shrimp, the latter of which correlated with an increase in the number of viable bacteria in the hemolymph. These results indicate that PmPPAE1 functions in the proPO system and is an important component in the shrimp immune system.