Study On The Regulatory Mechanism Of TBK1 On Inflammatory Signaling Pathway During Brucella Infection In Host Cells

Objective: the purpose of this study was to study the regulation of TBK1 on inflammatory signaling pathway during Brucella infection,and to explore the "means" of immune escape based on TBK1,so as to lay a foundation for further understanding of innate immune response and revealing the mechanism of intracellular parasitism of Brucella.Methods:(1)to explore the effect of Brucella infection on NF-κB signal pathway.Mouse macrophage RAW264.7 was infected with abortive Brucella S2308.0,4,12,24,48 h m RNA and protein were collected,and proinflammatory cytokines(IL-1,IL-6,TNF-α)and NF-κB signal pathway proteins(IKBα,p65,P-IKBα,P-P65)were detected relatively quantitatively.The THP-1 of human myeloid leukemia mononuclear cells infected with S2308 was sequenced by m RNA Human Gene Expression Microarray V3.0,the differential expression of visual genes in R language was detected,the TBK1 interactive genes were enriched,and the biological function of TBK1 was analyzed by GO and KEGG.(2)to investigate whether TBK1 is involved in the expression of pro-inflammatory cytokines induced by activated NF-κB signal pathway in Brucella infection macrophages in vitro.Si-TBK1 interference fragments were designed and synthesized and PCDNA3.1-TBK overexpression vectors were constructed and transfected into mouse macrophages RAW264.7 to establish a cell model for regulating TBK1 gene expression.S2308 was used to infect RAW264.7 of mouse macrophages transfected with si-TBK1 and PCDNA3.1-TBK1.12 h and 24 h m RNA and protein were collected to quantitatively detect pro-inflammatory cytokines(IL-1,IL-6,TNF-α)and NF-κB signaling pathway proteins(IKBα,p65,P-IKBα,P-P65),and to explore the effect of differential expression of TBK1 on intracellular survival of Brucella.(3)screening,identifying and verifying the Brucella proteins interacting with TBK1.Firstly,the yeast two-hybrid AD library and PGBKT7-TBK1 bait plasmid of Brucella secretory proteins were constructed to detect the self-activation of bait proteins and the cytotoxicity of yeast.Brucella secretory proteins interacting with TBK1 were screened by yeast co-transformation method.TBK1 and BMCO were modeled by Haddock2.4,Ligplot1.4.5,Py Mol2.2.0 and Prodigy.The protein binding sites were analyzed and the protein docking model was established.The fluorescence localization vectors of PDSRED2-C1-TBK1 and PDSRED2-C1-BMCO were constructed,and the cellular localization of TBK1 and BMCO was observed.The green fluorescent vector of PCDNA3.1-EGFP-TBK1 was constructed,and the co-localization of TBK1 and BMCO was observed.The functional vectors of PACGFP-TBK1 and PCMV-BMCO co-immunoprecipitation were constructed and co-transferred into 293 T cells.The interaction between TBK1 and BMCO was verified by co-immunoprecipitation.(4)to explore the biological significance of the interaction between TBK1 and BMCO,construct BMCO gene complementary plasmid,overlap extension PCR to fuse the upstream and downstream homologous arms of BMCO and kan resistance gene,construct Brucella BMCO gene deletion strain by kanamycin resistance replacement,and continuously culture for 15 generations to transfer p BBR1MCS-4-BMCO plasmid to the deleted strain which can stabilize genetic and biological characteristics.Mouse macrophage RAW264.7 was infected with S2308ΔBMCO,S2308 and S2308ΔBMCO::BMCO.12 h and 24 h m RNA and protein were collected.Proinflammatory cytokines(IL-1,IL-6,TNF-α),NF-κB signaling pathway proteins(IKBα,p65,P-IKBα,P-P65)and TBK1 were quantitatively detected.Mouse macrophage RAW264.7 was infected with plural infection(MOI)100.The survival numbers of parent strain,deletion strain and supplementary strain in cells were detected at different time points.Results:(1)after Brucella infection,NF-κB signal pathway was activated(P < 0.001),and the relative expression of proinflammatory cytokines(IL-1,IL-6,TNF-α)was significantly increased(P < 0.001),and this trend was most obvious at 12 h or 24 h.The transcriptome results showed that there were 71 differentially expressed m RNA(34 up-regulated and 37 down-regulated)during Brucella infection.The expression of TBK1 increased significantly during Brucella infection.The results of enrichment analysis showed that TBK1 participated in innate immune response,mediated the activation of immune signal pathway of DNA virus and RNA virus infection,and as an NF-κB activating factor,regulated NF-κB signal pathway and played an important role in the expression of downstream inflammatory cytokines.(2)the interference efficiency of interference fragment si RNA-TBK1-2 was 70%,and the expression efficiency of expression vector PCDNA3.1-TBK1 was 6 times.The cell model of TBK1 gene expression regulation was established successfully.During Brucella infection,TBK1 interference effectively decreased the expression of pro-inflammatory cytokines(IL-1,IL-6,TNF-α),inhibited the activity of NF-κB signal pathway,and increased the intracellular survival of Brucella(P < 0.001).Overexpression of TBK1 increased the activity of NF-κB signal pathway and promoted the expression of pro-inflammatory cytokines(IL-1,IL-6,TNF-α).The survival number of Brucella in cells was decreased(P < 0.001).(3)the yeast two-hybrid library of Brucella secreted protein was successfully constructed,and the PGBKT7-TBK1 bait protein had no self-activation and cytotoxicity.A Brucella secretory protein-BMCO which interacted with TBK1 was screened,identified and verified.The docking model of BK1 and BMCO was established by Haddock2.4.The dissociation constant is 1.4x10-10 and the binding energy is-13.4Kcal/mol.Both TBK1 and BMCO were located in the cytoplasm.The results of scanning laser copolymerization and immunoprecipitation showed that TBK1 and BMCO could combine stably and strongly with each other.(4)the BMCO gene deletion strain and complementary strain of Brucella with genetic stability were successfully constructed,which were named S2308 delta BMCO and S2308 delta BMCO:: BMCOS2308,respectively.When the cells were infected by different strains,the results showed that the expression of TBK1 was not affected by BMCO.BMCO could inhibit the host NF-κB signal pathway,reduce the expression of proinflammatory cytokines(IL-1,IL-6,TNF-α),and promote the intracellular survival of Brucella(P < 0.001).Conclusion: during the infection of macrophages in vitro,the secreted protein BMCO of Brucella can target TBK1,inhibit the activity of NF-κB signal pathway,reduce the expression of proinflammatory cytokines(IL-1,IL-6,TNF-α),increase the survival number of Brucella in cells,and promote the persistent infection of Brucella in cells.