Study On The Ligand Recognition And Signaling Pathway Of Two Membrane TLR5 Members In Grass Carp

Fish as lower vertebrate,which acquired immunity is not perfect,and their innate immunity plays important roles in the defense response to external stimuli and pathogen invasion.Toll-like receptors（TLRs）are important pattern recognition receptors（PRRs）in innate immunity,which can specifically recognize microbial-related molecular patterns（MAMPs）and trigger immune responses.TLR5 can recognize bacterial flagellin and induce inflammatory response,while study on ligand recognition and signaling transduction mechanism of fish TLR5 are still lacking.In this study,ligand recognition and signaling pathway of the CiTLR5a and CiTLR5b were investigated.The main results are as follows:1.CiTLR5a and CiTLR5b form heterodimer to sense flagellin,and form homodimer to sense dsRNA.CiTLR5a-GFP,CiTLR5b-GFP,CiTLR5a-RFP,CiTLR5b-RFP,CiTLR5a-HA and CiTLR5b-HA vectors were constructed and transferred into FHM cells,respectively.Confocal photography and Co-IP showed that CiTLR5a and CiTLR5b could form both homodimer and heterodimer.Subsequently,qRT-PCR,ELISA,pull-down and dual-luciferase reporter analyses showed that CiTLR5a and CiTLR5b could recognize flagellin and dsRNA,CiTLR5a and CiTLR5b form heterodimer to sense flagellin and improve NF-κB promoter activity.CiTLR5a form homodimer to sense dsRNA and weakly inhibit IFN response,while CiTLR5b homodimer sense dsRNA and promote IFN response.2.CiTLR5a and CiTLR5b play antagonistic roles in antiviral immune response.CiTLR5a-GFP,CiTLR5b-GFP and their small interfering RNA（si RNA）were transfected into juvenile grass carp（Ctenopharyngodon idella）and C.idella kidney（CIK）cells,respectively.CIK cells were infected with grass carp reovirus（GCRV）and Aeromonas hydrophila.The virus titer and the damage of virus for CIK cells were detected,respectively.Meanwhile,dual-luciferase reporter assay,plaque assay,TCID₅₀,virus titer,MTT assay,cell-level and individual-level interference,and immunoblotting assay showed that CiTLR5a and CiTLR5b play antagonistic roles in dsRNA recognition.CiTLR5a could weakly inhibit the antiviral response,while CiTLR5b can promote the antiviral response.3.CiTLR5a and CiTLR5b sense flagellin are conserved,while sense dsRNA with sequence and species differences.Firstly,the chimeras of CiTLR5a and CiTLR5b were constructed,and dual-luciferase reporter assay showed that extracellular domains of CiTLR5a and CiTLR5b play key roles in ligand recognition and signaling transduction.According to the crystal structure of zebrafish TLR5b and flagellin complex,two conserved sites of CiTLR5a and CiTLR5b were been changed,respectively.pull-down and dual-luciferase reporter analyses showed that both CiTLR5a and CiTLR5b mutants can reduce binding ability and signaling transduction for flagellin,which indicated that CiTLR5a,CiTLR5b and zebrafish TLR5b were highly conserved in flagellin recognition.The key dsRNA binding sites of CiTLR5a and CiTLR5b were been predicted and changed.pull-down and dual-luciferase reporter analyses showed that the binding ability of CITLR5A-G240A and CITLR5B-N547A for dsRNA were reduced.These results indicated that CiTLR5a and CiTLR5b have different dsRNA recognition locations.4.N-Glycosylation of CiTLR5a and CiTLR5b affect their immune modulating function.Immunoblotting detection showed that CiTLR5a and CiTLR5b bandswere larger than that their theoretical values.After treatment with Endo H,CiTLR5a and CiTLR5b were decreased to the theoretical values that 100 kDa.CiTLR5a and CiTLR5b were transfected into FHM cells and treated with tunicamycin（glycosyltransferase inhibitor）,and dual-luciferase reporter assay showed that functions of CiTLR5a and CiTLR5b were disappeared.The N-glycoylation sites of CiTLR5a and CiTLR5b were been predicted and site-directed mutated.Dual-luciferase reporter assay showed that CITLR5a-T101I could promote IFN1 promoter activity,while CiTLR5b-I99T decreased IFN1 promoter activity.The different N-glycosylation sites of CiTLR5a and CiTLR5b did not affect NF-κB promoter activity.pull-down analysis showed that the different N-glycosylation sites of CiTLR5a and CiTLR5b had no significant effect on the flagellin and dsRNA binding.5.Cyprinid fish TLR5 can sense dsRNA.The plasmidsof TLR5 that form gar,zebrafish,channel catfish,rainbow trout and human were constructed,which were transferred into FHM cells for pull-down with Fla C and poly（I:C）in acidic（p H 5.0）and neutral（p H 7.4）conditions,respectively.The results showed that only zebrafish TLR5a and TLR5b could recognize both poly（I:C）and Fla C and induce immune response,while TLR5 from other species only recognized Fla C.These results suggest that cyprinid TLR5（TLR5a and TLR5b）can recognize flagellin and dsRNA.Collectively,these results showed that CiTLR5a and CiTLR5b could recognize flagellin and dsRNA,which formed heterodimer to sense flagellin.CiTLR5a and CiTLR5b form homodimers to sense dsRNA and play antagonistic roles in antiviral immune response.We first systematacially study the ligand recognition and immune regulation mechanism of fish TLR5.These results will expand the understanding for immunomodulatory function of TLR and provide important theoretical basics for comparative immunological studies.