Molecular Basis Of Toll-like Receptor Mediated Antiviral Innate Immunity In The Pacific Oyster

The Pacific oyster(Crassostrea gigas)is one of important marine aquaculture species in China,while summer mortality events have been reported occasionally.The economic losses directly affect the sustainable development of Chinese aquaculture industry.It has been studied that the Ostreid Herpevirus-1(OsHV-1)is an important pathogen that causes the oysters' mortality.Therefore,in-depth study of the anti-virus immune defense mechanism of Pacific oyster is of great significance to the exploration of virus prevention.Toll-like receptor(TLR)is an important pattern recognition receptor(PRR)in recognizing and eliminating pathogens.The mechanism of TLR signaling pathway has been studied more intensively in vertebrates and is still in an exploratory stage in the study of invertebrates.It has been found that the TLR genes have obvious expansion in several invertebrates,and hundreds of TLR homologous(Crassostrea gigas,Capitella capitata,Strongylocentrotus purpuratus)are predicted in the genome,suggesting that the TLRs in invertebrates have functional differentiation.At present,the research on the TLR signaling pathway in molluscs is poor,and the results obtained are relatively simple and fragmented.Therefore,an in-depth study of the mechanism of key genes in the TLR pathway helps us to better understand the role of oyster TLR signaling pathways in the anti-viral innate immune defense process.In this study,molecular biology and cell biology methods and so on were used to investigate the mechanism of key genes in the oyster TLR signaling pathway.A large number of studies have shown that TLR recruits adaptor proteins through TIR-TIR binding in vertebrates.We first used the four TLR genes that were up-regulated in the outbreak period of OsHV-1 as the research objects,and screened proteins that might interact with them through the yeast two-hybrid library system.Our results show that only one of these four TLR proteins,CgTLR-27513,identified interacting proteins that containing TIR domain.Therefore,we use CgTLR-27513 gene as the main component in our subsequent experiments.By analyzing the function of the remaining three TLR potential interacting proteins,we found those predicted interacting proteins participate in cell migration,respiration and cytokines transport.Therefore,we speculate the oyster TLR genes are functional diversification.There are 3 potential interacting proteins containing TIR domain screened by CgTLR-27513.Two of them are MyD88,adaptor proteins that have been cloned and identified,and the other is a MyD88-like protein with only a TIR domain named after CgMyD88 s.We found that CgMyD88-1 and CgMyD88-2 can bind to each other to form homo-or heterodimerization to participate in CgTLR-27513-mediated antiviral immunity.In addition,CgMyD88-1 aggregated in the cytoplasm under stimulation with LPS and poly(I:C),whereas CgMyD88-2 condenses in the cell membrane and cytoplasm under both LPS and poly(I:C)stimulation.These results suggest that CgMyD88-1 and CgMyD88-2 are functional differences in TLR signaling pathway.Combined with the past data in laboratory,CgMyD88 s exhibited a decreased and then increased expression pattern after stimulation with OsHV-1 ?var,which is the opposite of the two CgMyD88.The result of co-immunoprecipitation showed that CgMyD88 s could only bind with CgTLR-27513 but not with two CgMyD88.After interference with CgMyD88 s expression,both CgMyD88 expressions were significantly upregulated,and CgMyD88 s not only failed to activate NF-?B transcription but also inhibited the activation of NF-?B by two CgMyD88 proteins.Therefore,we speculated that CgMyD88 s competes with two CgMyD88 proteins.Secondly,we cloned a member of IRAK family which acted as a downstream molecular of MyD88 genes and named it CgIRAK4 based on its structural features.CgIRAK4 mRNA was up-regulated after OsHV-1 ?var,Vibrio alginolyticus and poly(I:C)stimulation,and it was able to participate in the TLR-mediated immune process by binding to two CgMyD88 through death domain.However,CgIRAK4 is unable to activate the transcriptional activity of NF-?B,while it could inhibit the transcriptional activation of NF-?B by two CgMyD88 proteins.This indicates that CgIRAK4 is involved in the negative regulation of MyD88-dependent TLR signaling pathway.Finally,we first cloned the TBK1 and IKK? genes which participate in MyD88-independent signaling pathway(TLR-TRIF)in oysters.Both of them are significant up-regulated after OsHV-1 ?var,Vibrio alginolyticus and poly(I:C)stimulation.CgTBK1 is expressed in the cytoplasm and needs to bind to CgSTING to activate the transcriptional activity of IFN-?;CgIKK? can induce the activation of NF-?B and IFN-? transcription by itself,whereas the truncation only containing kinase domain of CgIKK? loses this activation.Although there have been no reports of MyD88-independent signaling pathways in invertebrates,there are TLR-TRIF downstream TBK1 and IKK? genes involved in the innate immune process.In summary,this study first proposed that the TLR genes of Pacific oyster may assist the antiviral natural immune process through cell migration and respiration.Set up the mechanism of the anti-viral innate immune TLR pathway of Pacific oysters involved in CgTLR-27513,CgMyD88-1,CgMyD88-2,CgMyD88 s and CgIRAK4.Clone the downstream genes of MyD88-independent signaling pathway,CgTBK1 and CgIKK?,and investigate their function in the innate immunity initially.These results enrich the understanding of the function of invertebrate TLR signaling pathways,providing a theoretical basis for in-depth exploration of invertebrate immune system.