The Mechanism Study On The Anti-virus Function Of Peroxiredoxin4(Prx4)in Kuruma Shrimp Marsupenaeus Japonicus

BackgroundInnate immunity is a universal and basic response of host to the infection of pathogenic microorganism.Great progress have been acquired on the understanding of the molecular mechanism of the innate immunity from the research on some invertebrate model organisms such as Drosophila and Nematodes.In recent years,the marine culture industry,which represented by shrimp culture,has developed rapidly.However,various diseases caused by pathogenic microorganisms have caused huge losses to these aquaculture industries.Therefore,it is important and necessary to study the innate immunity mechanism of shrimp to provide theoretical reference and technical support for disease control of the related industry.Scientific questions and significanceThe antioxidase system of aerobe,traditionally including the superoxide dismutase,peroxiredoxin and catalase,was important to the innate immune response of animals.Recent studies have shown that Peroxiredoxin(Prx4),a multifunctional,widely distributed and thiol-dependent antioxidase,participates in innate immune responses and inhibits viral replication in animals.However,the molecular mechanism of Prx4 in the anti-virus immunity of animals is still unknown.In this study,the secretion of Prx4 protein into the hemolymph upon WSSV infection was discovered,it could worked as a damage-associated molecular patterns(DAMPs)to promote the translocation of Dorsal,the homologous protein of NF-kappa B transcription factor,into the nucleus and initiate the expression of antimicrobial peptides(AMPs).These AMPs play an important role in the antiviral immunity of shrimp.This is the first identification of a damage-associated molecular pattern protein that plays a role in the antiviral immunity of shrimp,expanding the knowledge on anti-oxidative protein,and help to understand the antiviral immune mechanism of shrimp and develop anti-wssv drugs for the breeding practice.Results1.MjPrx4 acts as a DAMPs to inhibit viral replication via the Dorsal-AMPs pathwayWe found that WSSV infection promoted the up-regulated expression of MjPrx4 protein in the immune organs and secretion of MjPrx4 in the hemolymph of prawn.Injection of purified recombinant MjPrx4 promotes the translocation of nuclear factor kappa B(NF-?B)homologous protein,Dorsal,into the nucleus and induces the expression of related antimicrobial peptide(AMPs)genes.Knock down MjPrx4 by using RNA interference technology or neutralize the secretion of MjPrx4 by using MjPrx4 specific antibody in vivo can inhibit the transfer of Dorsal into the nucleus,thereby inhibiting the expression of MjPrx4-dependent AMPs.Injection of purified recombinant MjPrx4 protein has the opposite function.Knock down the MjPrx4-dependent AMPs in shrimps by RNA interference(RNAi)technology resulted in the up-regulated expression of viral iel and vp28 genes and the increase of viral replication copy.This study demonstrated for the first time that MjPrx4 can act as DAMPs to promote MjDorsal nucleation,thereby inducing the expression of AMPs and inhibiting the replication of viral WSSV.2.MjTrx promotes the anti-virus immunity of shrimp and enhance the antiviral ability of MJPrx4Firstly,the gene of electron donor for MjPrx4 in its enzymatic circulatory system in Marsupenaeus japonicus was cloned,which was named MjTrx.The full length of the gene is 518 bp,and its complete open reading frame contains 315 bp base pairs encoding 105 amino acids.Expression pattern analysis showed that the transcription level of MjTrx was significantly up-regulated in various tissues by WSSV infection in shrimp.In vitro enzymatic reaction by using purified recombinant protein of MjTrx and MjPrx4 showed,MjTrx could enhance the enzyme activity of MjPrx4.Overexpression of MjPrx,MjTrx,and MjPrx+MjTrx in prawn can inhibit WSSV replication,and the MjPrx+MjTrx treatment is more efficient on inhibiting the WSSV replication compared with the other two groups,indicating that MjTrx can promote the antiviral ability of MjPrx4.Knockdown the expression of MjTrx by RNAi resulted in the decrease of survival rate,but recruitment by injection the MjPrx,MjTrx,and MjPrx+AjTrx protein led to obvious increase of survival rate,which proved that MjTrx promoted the Anti-virus immunity of shrimp and facilitated the anti-vurs function of MjPrx 4InnovationsThe paper disclosed the molecular mechanism about how MjPrx 4 inhibite the amplification of WSSV in shrimp.The main innovations were mentioned as follows:(1)Peroxiredoxin4(Prx4)act as DAMPs to restrict WSSV amplification through activating Dorsal translocation and inducing antivirus peptides expression in shrimp MjTrx can promote the anti-viral innate immune function of shrimp and facilitate the anti-virus function of MjPrx 4 by restoring MjPrx4 enzyme activity.