Identification and functional analysis of a thioester-containing protein from Portunus trituberculatus reveals its involvement in the prophenoloxidase system, phagocytosis and AMP synthesis

Abstract

Thioester-containing proteins (TEPs) are an ancient superfamily of secreted effector proteins that perform essential roles in the innate immune response. Herein, a TEP gene, designated as PtTEP, was identified from the swimming crab Portunus trituberculatus. The open reading frame (ORF) of PtTEP was 4434 bp in length, encoded a polypeptide with 1478 amino acids containing the conserved sequence features of insect TEP. PtTEP was highly expressed in most immune-related tissues, such as intestine, gill and hepatopancreas, and the expression of PtTEP was significantly up-regulated in hemocytes after bacterial and fungal challenges. Compared to that in fertilized eggs, the transcripts of PtTEP were decreased obviously in the cleavage stage and followed by a significant up-regulation in the blastula, gastrula and heart-beating stages. Combined with the higher mRNAs in crab ovary, it is indicated that PtTEP be a maternal immune factor. RNA interference-mediated suppression of PtTEP could significantly enhance the expression of prophenoloxidase (proPO) associated genes (PtproPO and PtPPAF) and serine protease related genes (PtcSP1–3 and PtSPH) but inhibit the expression of PtLSZ and phagocytosis-related genes (PtMyosin and PtRab5). These results were further supported by the PO and lysozyme activities in hemolymph of the PtTEP-silenced crabs. We also observed that silencing of PtTEP reduced the expression of antimicrobial peptide genes (PtALF1–3, PtCrustin1 and PtCrustin3) and the genes involved in activation of the Toll and NF-κB pathways and additionally increased the mortality of Vibrio alginolyticus infected crabs. Taken together, our study suggests that PtTEP might function in crab innate immune defense via regulating the proPO-activating system, phagocytosis and AMP synthesis.