Antiviral And Antibacterial Mechanism Of Peroxiredoxin Ⅳ In Kuruma Shrimp Marsupenaeus Japonicus

BackgroundInnate immunity is a natural defense mechanism commonly possessed by organisms to resist the invasion of pathogenic microorganisms from outside,and is the basis of the host to protect itself.The invertebrates only possessed the innate immune system.In model organisms represented by drosophila and nematodes,the research on the innate immune system has made great progress during the past few decades.In recent years,the marine invertebrate aquaculture industry represented by prawns has developed rapidly,but various diseases caused by pathogenic microorganisms have brought huge losses to this industry,study on the innate immune mechanism of prawns can provide theoretical basis and technical support for disease prevention and control in related industries.Scientific issues and research significancePeroxiredoxin IV（Prx4）,as a multifunctional and widely distributed thioldependent antioxidant enzyme,plays an important role in the innate immune response of organisms.Our previous studies have shown that MjPrx4 can inhibit the replication of White spot syndrome virus（WSSV）in Marsupenaeus japonicus,but its antiviral mechanism is unclear.In addition,the function and mechanism of MjPrx4 in the antibacterial immunity of Marsupenaeus japonicus is also unknown.In-depth exploration of the function and mechanism of MjPrx4 in the immune response of shrimp can provide help for understanding the function of Prx4 protein in the crustaceans and developing the control strategy for disease control.Results1.MjPrx4 restrained WSSV replication in shrimp by interacting with IE1 protein and inhibit the DNA binding activity of IE1.In this study,we found that MjPrx4 can enter the cellular nucleus after white spot syndrome virus infection and interact with WSSV immediate-early gene 1（IE1）product.By competitively binding with the DNA binding site of IE1 protein and inhibiting its DNA binding activity,the replication of the virus in the shrimp was inhibited.In addition,mortality experiments,co-immunoprecipitation（CoImmunoprecipitation,Co-IP）,electrophoretic mobility shift assay（EMSA）and mutant experiments showed that the antiviral activity of MjPrx4 depends on the MjPrx4 dimer formation,and three cysteines in the DNA binding site of IE1 are necessary for the interaction of the two proteins.We further found that the dimer formation and nuclear translocation of MjPrx4 was induced by the increase of H2O2 concentration caused by viral infection,and N-Acetyl-L-cysteine（NAC）treatment inhibited the dimer formation and nuclear translocation of MjPrx4,caused the increase of virus titer in shrimp.2.MjPrx4 participated in the antibacterial immune response of shrimp through activating the JAK/STAT pathway.In this study,we found that the expression of MjPrx4 in shrimp increased significantly after bacterial infection,especially in the extracellular environment.After MjPrx4 RNA interference in shrimp,the bacterial clearance ability of shrimp decreased obviously compared with that in control group and the mortality of the shrimp increased significantly.In addition,the phosphorylation and nuclear translocation of the transcription factor STAT,and the expression of five antimicrobial peptides（AMP）downstream of STAT were also inhibited.Furthermore,in vivo interference of Dome,MjPrx4 antibody blocking and external injection of MjPrx4 or its mutant protein proved that the extracellular MjPrx4 promotes the nuclear translocation of STAT and the expression of antimicrobial peptides through the Dome receptor.The above experimental results indicated that the MjPrx4 secreted into the hemolymph upon the bacterial infection,and then activate the JAK/STAT pathway through the Dome receptor,and promoted the expression of antibacterial peptides to exert anti-bacterial immune function.Research innovation and summaryThis thesis mainly studies the anti-WSSV and anti-bacterial immune function of MjPrx4 and clarifies its molecular mechanism in shrimp.The innovation points are summarized as follows:1.Our research reveals for the first time a new mechanism by which the shrimp Prx4 protein interacts with viral protein IE1 to inhibit viral replication.Studies have found that WSSV infection can promote MjPrx4 enter the nucleus,and interact with the IE1 of WSSV in a dimer-dependent manner.By binding to the DNA binding site of WSSV IE1 and inhibiting its DNA binding activity,MjPrx4 paricipated in the antivirus immunity of shrimp.2.In shrimp,the bacterial infection resulted in the increased expression of MjPrx4 in the hemolymph,then,activates the JAK/STAT pathway through the Dome receptor in the cytomembrane,promote the phosphorylation of the transcription factor STAT and enter the nucleus,and induces the expression of antibacterial peptides to participated in the antibacterial immunity of animal.