Functional Study Of Prohibitin And Key Molecules In JAK/STAT Signal Pathway In Crustacean Immune Responses

Crustaceans are important economic creatures, and crustaceans farming and fishing accounts for twenty percent of the world economy aquatic organisms. However, in recent years, aquaculture of aquatic diseases caused massive economic losses. The study of the crustacean innate immunity necessary, it was benefit for prevention and control of aquatic diseases, as well as the mechanism of immunity.In the present study, we identified a prohibitin from red swamp crayfish and found that it can inhibit WSSV replication. We have studied the mechanisms of signal transduction and transcriptional activity signaling pathways, and we explore the role of STAT signaling pathway in bacteria immunity. We have cloned a crustin which with a transmembrane domain, and found it involved in phagocytosis.1. Prohibitin1prevents infection of WSSV in red swamp crayfishProhibitins (PHBs) are ubiquitously expressed conserved proteins in eukaryotes that are associated with apoptosis, cancer formation, aging, stress responses, cell proliferation, and immune regulation. However, the function of PHBs in crustacean immunity remains unknown. In the present study, we identified a PHB in red swamp crayfish Procambarus clarkii, which was designated as PcPHB1. PcPHB1was widely distributed in several tissues and its expression was significantly upregulated by white spot syndrome virus (WSSV) challenge at the mRNA level and the protein level. These observations prompted us to investigate the role of PcPHB1in the crayfish antiviral response. Recombinant PcPHB1significantly reduced the amount of WSSV in crayfish and the mortality of WSSV-infected crayfish. The quantity of WSSV in PcPHB1knockdown crayfish was increased compared with the controls. The effects of RNA silencing were rescued by rPcPHB1re-injection. We further confirmed the interaction of PcPHB1with the WSSV envelope proteins VP28, VP26, and VP24using pull-down and far-Western overlay assays. Finally, we observed that the colloidal gold-labeled PcPHB1was located on the outer surface of the WSSV, which suggests that PcPHB1specifically binds to the envelope proteins of WSSV. VP28, VP26, and VP24are structural envelope proteins and are essential for attachment and entry into crayfish cells. Therefore, PcPHB1exerts its anti-WSSV effect by binding to VP28, VP26, and VP24, preventing viral infection. This study is the first report on the antiviral function of PHB in the innate immune system of crustaceans.2. JAK/STAT signaling pathway associated with antibacterial response in shrimpJAK/STAT pathway was reported involved in immune response of anti-bacterial and anti-viral immune responses. The suppressor of cytokine signaling proteins (SOCS) is a class of STAT induced STAT inhibitor (SSI), which negatively regulates the JAK/STAT signaling pathway. In shrimp, STAT was up-regulated expression in challenge with ploy IC and PGN. WSSV could induce the increase expression of STAT, and it induced the phosphorylation of STAT in shrimp. When the STAT was phosphorylation, it would translocation to the nuclear of cell and regulated the expression of effectors. However, the molecular mechanisms of STAT involved in bacterial immune are not clear in shrimp. In the present study, we found that the cell wall molecule of Vibrio anguillarum could induce the phosphorylation of STAT. SOCSs are feedback inhibitors of STAT pathway, SOCS1and SOCS3reported inhibiting the phosphorylation of STAT, and SOCS2had been reported to participate in NF-κB signaling pathways. Although there have been reports that SOCS2can be up-regulatedby ploy IC and PGN, which indicating that SOCS2may be involved in STAT phosphorylation process. However, there no report showed that SOCS2could inhibit the phosphorylation of STAT. In the present study, we found LPS could induce the phosphorylation of STAT. The special lectin MjIL-CT-L, which with IL similar domain, could bind to LPS and the ILR domain of Dome. And when Dome was knocked down, the phosphorylation of STAT was inhibited in challenge with LPS. SOCS2is able to feedback inhibition the process of STAT phosphorylation.We screened out some AMPs, which were induced expression by LPS specially. When STAT was knocked down in vivo, the bacterial clearance level was lower than the control, and the mortality of shrimp infected with bacteria was increase comparing with control. When SOCS2was knocked down in vivo, the bacterial clearance level was higher than the control, and the mortality of shrimp challenge with bacteria was decrease. These results indicate that STAT pathway involved in antibacterial immune response by regulating the expression of antimicrobial peptides in shrimp.3. A novel crustin from Marsupenaeus japonicus promotes hemocyte phagocytosisThe crustins, one kind of antimicrobial peptides (AMPs) from crustacean with a whey acidic protein (WAP) domain have functions in protease inhibition and antimicrobial activity. In the percent study, a novel crustin with a single WAP domain and a transmembrane region was found in kurume shrimp, Marsupenaeus japonicus, and designated as MjMCru. Quantitative real-time PCR (qRT-PCR) analysis showed that MjMCru was mainly expressed in heart, gills, stomach and, and low level expression in hemocytes and hepatopancreas. The expression level of MjMCru was up-regulated in hemocytes challenge with bacteria. Injection of WAP peptide could increase the survival rate of shrimp infected with V. anguillarum and S. aureus. Further study found that MjMCru could bind to bacteria by binding to the bacteria cell wall molecules such as LPS, PGN and LTA. MjMCru (WAP) could increase the phagocytic rate of hemocytes in shrimp infected with V. anguillarum and S. aureus. These results suggested that MjMCru had an important role in antibacterial immunity of shrimp.