| Brucellosis is a zoonoticinfectious disease,in which patients with brucellosis present some manifestations such as undulant fever,arthritis,endocarditis and meningitis,and animals infected by Brucella often occur abortion and infertility,which causes huge economiclosses and a major publichealth burden.The Outer membrane protein(Omp)of Brucella plays an important role in bacterial infection and pathogenicity.According to the molecular weight,Omps can be divided into three groups and 6 Omps are more researched at present.Brucella Type IV secretion system(T4SS)is a multi-protein transmembrane complex encoded by vir B operon and regulated by the same promoter,which plays an important role in mediating bacterial intracellular survival and manipulating host innate and adaptive immune responses.The effect of T4 SS is mainly realized by effector proteins secreted by itself,and a total of 15 effector proteins depending on T4 SS have been identified up to now.Brucella can cause immunosuppression in the body,but the effect and mechanism of Brucella Omps and T4 SS effector proteins on the inhibition of innate immunity are still unclear.In this study,Omps and T4 SS effector proteins that can significantly inhibit interferon-β(IFN-β)production were screened firstly.Then,the effects of the screened proteins for the transcription of IFN-β and downstream interferon stimulation genes(ISGs)induced by viruses were examined.Following,the production of these proteins for DNA virus-induced IFN-β and the effects of these proteins on cGAS-STING signaling pathway were detected respectively.After that,the target molecules and their modes of action in the signaling pathway were determined,and their interactions with the target molecules in the signaling pathway were detected.Thereafter,proteins that directly interact with each other were truncated,and then the binding domain of the interaction between these truncations and the target molecules in the signaling pathway was detected.The effects of the outer membrane protein and the T4 SS effector protein truncations on the transcription and production of IFN-β induced by DNA viruses were then examined to identify the functional binding domain that inhibits IFN-βproduction.Finally,key amino acid sites between the deletion and mutant functional binding domains were detected on the effects of deletion and mutant for IFN-βtranscription,production and cGAS-STING signaling pathway.The purpose of this study was to explore the effect and mechanism of Brucella Omps and T4 SS effector proteins in inhibiting DNA virus-induced IFN-β production,in order to provide theoretical basis for further exploring the mechanism of Brucella immunosuppression.These results were as follows.1.Brucella Omp25,Bsp B and RicA inhibit ifn-β promoter activityUsing inactivated Brucella abortus genomicDNA as a template,the lentiviral expression vectors for the major Omps and T4 SS effector proteins of B.abortus were constructed,and the results of enzyme digestion and sequencing identification showed that the above each protein that were lentiviral expression vector were constructed successfully.The lentivirus expression plasmids with PMD2.G and ps PAX2 were co-transfected into HEK-293 T cells,then RAW264.7 cells were infected by collected lentiviral.The results showed that Omps and T4 SS effector proteins were expressed in RAW264.7 cells,and Brucella Omp25,Bsp B and RicA were screened to significantly inhibit the activity of the ifn-β promoter(P < 0.01).2.Mechanism of Omp25 interacts with cGAS to degrade cGAS and inhibits IFN-β production through the ubiquitin proteasome pathwayLv-Omp25 significantly inhibited the transcription and the expression of IFN-β and the transcription of ISGs induced by DNA viruses(P < 0.01).Lv-Omp25 could significantly promote the replication of PRV and HSV-1.Lv-Omp25 could significantly inhibit the phosphorylation of STING and IRF3 and the entry of p-IRF3 into the nucleus(P< 0.01),and we found that Lv-Omp25 degraded cGAS through ubiquitin proteasome pathway,and inhibited the enzyme activity of cGAS significantly(P < 0.01).Further experiments showed that Omp25 interacted with cGAS,and Omp25 and cGAS were truncated.The results showed that Omp25(161-184 aa)and cGAS(161-522 aa)were the key binding domains of interaction.Omp25(161-184 aa)was a key binding domain that inhibits DNA virus-induced transcription and expression of IFN-β.After deletion of aa161-184,the inhibitory effect of Omp25Δ161-184 on DNA virus-induced IFN-βtranscription,expression and cGAS activity were significantly attenuated(P < 0.05),the inhibitory effect of Omp25Δ161-184 on HSV-1-induced expression of p-STING,p-IRF3 and cGAS were significantly attenuated(P < 0.05).Omp25-5M was mutanted at arginine176,180 and tyrosine 179,181,184 in the aa 161-184 region,the results showed that the inhibitory effect of Omp25-5M on DNA virus-induced IFN-β transcription and expression,and cGAS activity was distinctly weakened(P < 0.05),and the inhibitory effect on p-STING,p-IRF3 and cGAS expression was significantly weakened induced by HSV-1(P< 0.05).3.Mechanism of RicA interacts with STING to degrade STING and inhibits IFN-β production via the autophagy-lysosomal pathwayLv-RicA significantly inhibited the transcription and the production of IFN-β and the transcription of ISGs induced by DNA viruses(P < 0.01).Lv-RicA increased the DNA copy number of PRV and HSV-1(P < 0.01),and Lv-RicA could significantly promote the replication of PRV and HSV-1(P < 0.01).Lv-RicA significantly inhibited the phosporylation of TBK1 and IRF3 and degraded STING via the autophagy-lysosomal pathway.Further studies found that RicA could interact with STING.While RicA and STING were truncated respectively,the results showed that RicA(1-88 aa)and STING(1-153 aa)were the key binding regions for their interaction,and RicA(1-88 aa)could significantly inhibit the transcription and production of IFN-β(P < 0.01).After RicA 1-88 aa were deleted segmentally,the results showed that the RicAΔ1-20 for the activity of ifn-βpromoter was decreased significantly(P < 0.05),the inhibition of IFN-β transcription and expression level induced by DNA viruses were decreased significantly(P < 0.05),and the inhibition of TBK1 and IRF3 phosphorylation induced by HSV-1 were weakened(P <0.05).RicA-3M was mutanted at tyrosine 4,6 and arginine 16 in the aa 1-20 region,the results showed that the inhibitory effect of RicA-3M on DNA virus-induced IFN-βtranscription and expression was distinctly weakened(P < 0.05),and the inhibitory effect on p-TBK1 and p-IRF3 was significantly weakened induced by HSV-1(P < 0.05).4.Mechanism of Bsp B and TBK1 competitively bind IRF3 to reduce TBK1 phosphorylation of IRF3 and nucleation of p-IRF3,and inhibit IFN-β productionLv-Bsp B significantly inhibited the transcription and the production of IFN-β and the transcription of ISGs induced by DNA viruses(P < 0.01).Lv-Bsp B increased the DNA copy number of PRV and HSV-1(P < 0.01).Further studies showed that the target site of Bsp B on cGAS-STING was IRF3.Lv-Bsp B significantly inhibited DNA virus-induced IRF3 phosphorylation and p-IRF3 nuclear transfer(P < 0.01).Further experiments showed that Bsp B interacted with IRF3.Truncated Bsp B and IRF3 showed that Bsp B(156-187 aa)and IRF3(198-427 aa)were the key binding domains for their interaction,and Bsp B(156-187 aa)significantly inhibited the transcription and expression of IFN-β induced by DNA viruses(P < 0.01).After deletion of Bsp B(156-187 aa),the results showed that the inhibitory effect of Bsp BΔ156-187 on the transcription and production of IFN-β induced by DNA virus were attenuated(P < 0.05),Bsp BΔ156-187 inhibited the phosphorylation of IRF3 induced by HSV-1(P < 0.05).Bsp B competed with TBK1 to bind to IRF3,resulting in a decrease in the phosphorylation of IRF3 by TBK1,and the nuclear transcription factor IRF3.While Bsp B-4M was mutanted at arginine 179,181,183 and 184 in the 156-187 region,the results showed that the inhibitory effect of Bsp B-4M on DNA virus-induced IFN-β transcription and expression was distinctly weakened(P < 0.05),and the inhibitory effect on IRF3 phosphorylation and p-IRF3 nuclear entry was significantly weakened induced by HSV-1(P < 0.05).In this study,lentiviral expression vectors of the main Omps and T4SS-dependent effector proteins of Brucella were successfully constructed,and from which Omp25,Bsp B and RicA that significantly inhibited the activity of the ifn-β promoter were screened.It was found that Omp25(161-184 aa),RicA(1-20 aa),and Bsp B(156-187 aa)were functional binding domains of Brucella Omp25,RicA,and Bsp B to inhibit IFN-β production.The results elucidate the function and regulatory mechanism of Brucella Omp25,RicA and Bsp B in inhibiting IFN-β production induced by DNA viruses,and provide theoretical basis for further research on the pathogenesis of Brucella. |
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