The Function Analysis Of Two Peroxiredoxin Subfamilies In Innate Immunity Of Kuruma Shrimp Marsupenaeus Japonicus

BackgroundInnate immunity is the inherent and fundamental defense mechanism against the invasion of pathogenic microorganisms for animals. Innate immunity is also the basis of adaptive immunity activation. Invertebrates, only persisting the innate immune system, are satisfactory experimental animals for the mechanism research of innate immunity and have made great progress in some model organisms such as Drosophila and Nematodes. Recently, the marine culture industry, which represented by shrimp culture, has developed rapidly, but suffered great losses because of the outbreak of shrimp diseases. Therefore, it is important and necessary to study the innate immunity mechanism of shrimp to provide theoretical reference and technical support for shrimp disease control.Questions and significanceThe antioxidase system of aerobe, traditionally including the superoxide dismutase and catalase, is the important part of innate immune systems of animals. Recent studies have shown that Peroxiredoxin (Prx), a multifunctional, widely distributed and thiol-dependent antioxidase, may be involved in the innate immune response of animals. However, the definitive function and molecular mechanism of Prx in the innate immune response of animals is still unknown. In this study, the kuruma shrimp Marsupenaeus japonicus was selected as the experimental animal, the function and mechanism of Prxl subfamily protein in antiviral immunity and 1-Cys Prx6 subfamily protein in anti-bacterial immunity of shrimp was studied. Our results will help to understand not only the function of Prx, but also the function of antioxidase system in innate immunity of animals. The results may also provide theoretical reference for shrimp disease control.Results1. MjPrxl enhance WSSV replication through mediating NF-κB homolog MjDorsal dimerization and promoting the activity of MjDorsal binding to WSSV iel promoterFirstly, the expression of six subfamily members of MjPrx in shrimp was studied at various time post-challenge with white spot syndrome virus (WSSV). The MjPrxl showed the most significant up-regulated expression patterns, so it was selected for further study. RNA interference revealed that Mjprxl knockdown could elevate the survival rate of WSSV-infected shrimps and depress the replication of WSSV, while the injection of exogenous MjPrxl recombinant protein could promote WSSV replication. Immunocytochemistry and Chromatin Immunoprecipitation experiments revealed that WSSV challgenge induced the nuclear translocation of MjDorsal (NF-κB homolog), and its activity binding to the promoter of WSSV iel gene, but these effects were inhibited in Mjprxl knockdown shrimp. Co-immunoprecipitation assay proved that MjPrxl could interact with MjDorsal and formed heterodimer in vivo of shrimp, this interaction was promoted by WSSV infection. Furthermore, MjPrxl could interact with MjDorsalRHD overexpressed in Helicoverpa armigera epidermal cell line, and mediated the formation of disulfide bond-linked MjDorsalRHD homodimers. Meanwhile, the site-directed mutagenesis of MjPrxl and Pulldown assay indicated the Cys51 residue of MjPrxl is critical for the interaction of MjPrxl and MjDorsal and important for the function of MjPrxl promoting WSSV replication. These results indicated that MjPrxl could promote WSSV replication in vivo of shrimp through mediating the dimerization of MjDorsal and promoting its nuclear translocation and activity binding to WSSV iel promoter in shrimp.2.1-Cys Prx6 functioned as the regulator of antimicrobial peptide expression through maintaining redox homeostasis, protected shrimp from bacteria infectionIn Marsupenaeus japonicus, the expression of MjPrx6 and H2O2 concentration in vivo of shrimp was induced by Vibrio anguillarum infection. Knockdown of Mjprx6 engendered the increased mortality of shrimps and the decreased bacterial clearance ability, which indicated an important role of MjPrx6 in shrimp anti-bacterial innate immunity. Furthermore, Mjprx6 knockdown induced the abnormal H2O2 elevation and the inhibited expression of some antimicrobial peptide (AMP) gene. These phenomenons could be released by the injection of antioxidant NAC into shrimp. Then exogenous gradient H2O2 were injected into shrimp to mimic the different H2O2 concentrations that caused by vibrio infection or Mjprx6 knockdown. The results showed that:low H2O2 concentration promoted AMP gene expression, while the high H2O2 concentration inhibited AMP gene expression and induced the trascription of Mjprx6 gene. These results indicated that H2O2 could bilaterally regulate the expression of AMP gene in a dose-dependent manner, and the oxidative stress can induce the up-regulation of Mjprx6 gene. Then, the recruitment experiment in Mjprx6 interference shrimps revealed that the wild-type MjPrx6 recombinant protein could decreased the abnormal H2O2 concentration induced by Mjprx6 interference, and restore the imperfect bacterial clearance ability and the inhibited AMP gene expression caused by Mjprx6 knockdown. However, the mutant MjPrx6C44S protein without peroxidase activity showed no effect. RNA interference of AMP gene and bacterial clearance test further showed that AMP regulated by MjPrx6 played an important role in anti-bacterial innate immunity. These results showed that MjPrx6 could function as antioxidase, prevent the massive H2O2 generation induced by bacteria infection, maintain H2O2 concentration at a modest level that promote AMP gene expression.InnovationsThis research mainly investigated the function and molecular mechanism of two subfamily members of Prx protein in the innate immunity of shrimp Marsupenaeus japonicus.The main innovations were mentioned as follows.(1) We firstly reported Prxl interacted with NF-κB homolog MjDorsal, promoted its nuclear translocation and its activity binding to WSSV iel promoter through mediating the formation of its disulfide bond-linked homodimer, and promoted WSSV virus replication in vivo of shrimp.(2) It is the first report that H2O2 functioning as the signal could bilaterally regulate AMP gene expression in the dose-dependent manner.(3) We verified 1-Cys Prx6 could regulate AMP gene expression through regulating H2O2 level in the peroxidase activity-dependent manner.