Function And Mechanisms Of Interferon Regulatory Factors IRF1,IRF3,and IRF7 In Antibacterial Innate Immunity Of Trachinotus Ovatus

Interferon regulatory factors(IRFs)are crucial transcription factors in modulating transcription of interferons(IFNs).IRFs directly binds to the type I IFNs virus‐enhancer elements to regulate the expression of Type I IFNs and other genes in the IFN pathway.IRFs have important roles not only in immune response,antiviral,and antibacterial,but also in cell proliferation and apoptosis.IRFs play a crucial part in antiviral host defence,but the role of IRFs in bacterial infection remains poorly understood.In this study,we cloned IRF1,IRF3 and IRF7 from golden pompano(Trichinous ovatus)and investigated their functions and mechanisms in antibacterial.The results are as follows.TroIRF1,an important member of the IRF family.TroIRF1 has an open reading frame(ORF)of 927 bp that encodes a protein of 309 amino acids with a calculated molecular weight of 35 k Da.Multiple amino acid alignments of TroIRF1 shows highest identity with Siniperca chuatsi IRF1’s sequence(78.41%).The constructed phylogenetic tree revealed that TroIRF1 was more closely related to IRF1 of S.chuatsi.TroIRF1 expressed constitutively in fish tissues under normal physiological conditions,with high levels in gill,spleen,and head kidney.Following the V.harveyi challenge,TroIRF1 transcripts were up-regulated in spleen,head-kidney,intestine,and gill.In vivo,TroIRF1 over-expression promoted the fish to inhibit the replication of V.harveyi.And TroIRF1 knockdown led to decreased bacterial clearance in fish tissue.Furthermore,over-expression of TroIRF1 enhanced macrophage activation and increased the production of interferon a3(Tro IFNa3).TroIRF1 directly binds to Tro IFNa3 promoter,these are dependent on three conserved amino acids of the N-terminal DNA-binding domain helix α3 motif.TroIRF1 works in concert with My D88 to activate Tro IFNa3 promoter.As a transcription factor with constitutively nuclear retention,TroIRF1 contains two nuclear localization signals,both essential for the nuclear localization of TroIRF1 protein.TroIRF3,an important member of the IRF3 subfamily.The ORF of TroIRF3 is 1398 bp,which encodes 466 amino acid residues,and the molecular weight is 51 k Da.Multiple amino acid alignments of TroIRF3 show highest identity with Seriola lalandi dorsalis IRF3’s sequence(85.95%).The constructed phylogenetic tree revealed that TroIRF3 was more closely related to IRF3 of S.lalandi dorsalis.TroIRF3 expressed constitutively in fish tissues under normal physiological conditions,with high levels in gill,liver,and muscle.Following the V.harveyi challenge,TroIRF3 transcripts were up-regulated in spleen,head-kidney,intestine,and gill.In vivo,TroIRF3 overexpression promoted the fish to inhibit the replication of V.harveyi.And TroIRF3 knockdown led to decreased bacterial clearance in fish tissue.Furthermore,overexpression of TroIRF3 enhanced macrophage activation and increased the production of Tro IFNa3.TroIRF3 works in concert with My D88 to activate Tro IFNa3.Accurate cellular localization plays a crucial role in the effective function of most signaling proteins,and nuclear trafficking is central to the function of transcription factors.V.harveyi,E.tarda,and S.agalactiae induced TroIRF3 translocation to the nucleus.And we identified a bipartite nuclear localization signal(NLS)in TroIRF3,with two interdependent basic clusters separated by a 7-aa linker.TroIRF7,another important member of the IRF3 subfamily.The ORF of TroIRF7 is 1320 bp,which encodes 440 amino acid residues,and the molecular weight is 50 k Da.Multiple amino acid alignments of TroIRF7 shows highest identity with Epinephelus coioide IRF7’s sequence(85.18%).The constructed phylogenetic tree revealed that TroIRF7 was more closely related to IRF7 of E.coioide.TroIRF7 expressed constitutively in fish tissues under normal physiological conditions,with high levels in gill,spleen,head kidney,skin,and intestine.Following the V.harveyi challenge,TroIRF7 transcripts were up-regulated in spleen,head-kidney,intestine,and gill.And TroIRF7 translocated into nucleus from cytoplasm after V.harveyi stimulate.In vivo,TroIRF7 over-expression promoted the fish to inhibit the replication of V.harveyi.And TroIRF7 knockdown led to decreased bacterial clearance in fish tissue.Furthermore,over-expression of TroIRF7 enhanced macrophage activation and increased the production of Tro IFNa3.Analysing antibacterial functions and mechanisms of TroIRF1,TroIRF3 and TroIRF7 not only helps to further the current understanding of functions and mechanisms of fish IRFs,but also contributes to the understanding the immune system of golden pompano and providing safe and efficient avenues for protection against pathogen infection.