| Objective:The plague is a zoonotic disease caused by Yersinia pestis,which is a powerful infectious disease of natural foci.Yersinia is a short gram-negative bacillus.The type ? secretion system(T3SS)is an important virulence mechanism commonly found in gram-negative bacteria.When the bacteria infect host cells,T3SS is encoded by pYV or pCD virulence plasmids,virulence effector protein(Yops protein)secreted into host cells by a needle-like injection body structure.there are six known effector proteins:YopH,YopE,YopM,YopJ/P,YpkA and YopT.Yersinia pneumoniae,a gram-negative bacterial pathogen delivers six effector proteins into the host cell.By interfering the innate immune function of host cells,including inhibiting the host's inflammatory response,escaping non-specific immune protection,and resulting in escaping the phagocytosis and killing of phagocytes,which can continue to survive and proliferate in the host body,and continue to infect the host.One of the effectors,YopT,is an effective cysteine protease,which can cause the destruction of the actin cytoskeleton,and inhibiting the phagocytosis of pathogens.However,the mechanism of escape innate immunity and the pathogenesis of plague need to be further studied.Methods:1.Yeast two-hybrid technique was used to screen the protein interacting with the effector protein YopT of Yersinia pestis T3SS;the interaction between YopT protein and RIG-I protein was confirmed by co-immunoprecipitation;and the co-localization of YopT and RIG-I was confirmed by immunofluorescence.2.Using double luciferase reporter gene assay,it was confirmed that YopT inhibited the activation of NF-kappa B and IRF3 mediated by RIG-I in a dose-dependent manner;And that YopE and YopM did not affect the activation of NF-kappa B and IRF3 mediated by RIG-I.3.Double luciferase reporter gene assay was used to determine that YopT inhibited the activation of NF-? B or IRF3 induced by poly;And that YopT did not affect the activation of NF-? B or IRF3 mediated by MAVS and TBK1.4.Western blotting was used to determine that YopT down-regulated the expression of RIG-I protein in a dose-dependent manner,and that YopE,YopJ,YopM had no effect on the expression of RIG-I protein;while Western blotting was used to determine that YopT could shorten the half-life of RIG-I protein;Western blotting was used to determine the down-regulation of TBK1 phosphorylation and RIG-I protein expression after infection with wild(WT)strain and replicator strain(? YopT/YopT)of Yersinia pestis;And realtime fluorescence PCR assay was used to determine the down-regulated levels of IFN-?and IL-6 mRNA after infection with wild Yersinia pestis strain(WT)and replicator strain(? YopT/YopT).5.Western blot was used to determine that cysteine protease YopT could not cleave RIG-I protein;immunoprecipitation method was used to determine that YopT could increase the ubiquitination level of extraneous Flag-RIG-?;immunoprecipitation method was used to determine that YopT increased the ubiquitination level of RIG-? through lysine connection at position 48;immunoprecipitation method was used to determine that YopT could increase the ubiquitination level of endogenous RIG-I.Results:part ?:the interaction between RIG-I and YopT:In HEK293T cells co-transfected with Myc-YopT,Flag-Vector or Flag-RIG-I,it was found that Myc-YopT,was detected in the immunoprecipitation mixture of Flag-RIG-I co-transfected cell lysates incubated with Flag agarose magnetic beads,but no Myc-YopT protein was detected in Flag-Vector co-transfected cell lysates;In HEK293T cells cotransfected with Myc-YopE,Flag-Vector or Flag-RIG-I,it was found that Myc-YopE was not detected in the immunoprecipitation mixture of Flag-Vector or Flag-RIG-I cotransfected cell lysates incubated with Flag agarose magnetic beads;In HEK293T cells co-transfected with Myc-YopM,Flag-Vector or Flag-RIG-I,it was found that MycYopM was not detected in the immunoprecipitation mixture of Flag-Vector or Flag-RIG-I co-transfected cell lysates incubated with Flag agarose magnetic beads;In the HEK293T cells co-transfected with Myc-RIG-I and Flag-YopE,Falg-YopM,Flag-YopT,Flag-Vector,it was found that only the lysates of Flag-YopT co-transfected with Flag agarose magnetic beads were detected in the immunoprecipitation mixture of Myc-RIG-I;In the RAW264.7 cells co-transfected with Myc-vector,Myc-YopT,and it was found that Myc-YopT and RIG-I were co-located.Part ?:YopT inhibits RIG-?-mediated activation of NF-kappa B and IRF3:In HEK293T cells co-transfected with pGL3-IRF3-luc/UAS-Luc and Myc-vector or Myc-RIG-I and Flag-vector,Flag-YopT,Flag-YopE,Flag-YopM plasmid respectively,it was found that overtransfection of Flag-YopT decreased the activity of IRF3 luciferase compared with the control group cotransfected with Flag-vector;In HEK293T cells cotransfected with pGL3-NF-kappa B-Luc and Myc-vector or Myc-RIG-I with Flagvector,Flag-YopT,Flag-YopE,Flag-YopM plasmid respectively,it was found that overtransfection of Flag-YopT decreased the luciferase activity of NF-kappa B compared with overtransferred Flag-vector control group;In HEK293T cells co-transfected with pGL3-IRF3-luc/UAS-Luc and Flag-vector or Flag-RIG-I with different doses of MycYopT,it was found that YopT decreased IRF3 luciferase activity in a dose-dependent manner;While in HEK293T cells co-transfected with pGL3-NF-? B-Luc and Flag-vector or Flag-RIG-I with different doses of Myc-YopT,YopT decreased NF-?B luciferase activity in a dose-dependent manner;In HEK293T cells co-transfected with pGL3-IRF3?luc/UAS-Luc and poly(poly C)and Flag-vectoFlag-YopT,Flag-YopE,Flag-YopM plasmid respectively,it was found that overtransfection of Flag-YopT decreased IRF3 luciferase activity compared with overtransferred Flag-vector control group;In HEK293T cells co-transfected with pGL3-NF-kappa B-Luc and poly(poly)and Flagvector,Flag-YopT,Flag-YopE,Flag-YopM plasmid respectively,it was found that overtransfection of Flag-YopT decreased the luciferase activity of NF-kappa B compared with the overtransferred Flag-vector control group.Part ?:YopT inhibits the activation of NF-?B and IRF3 induced by poly(I:C).In HEK293T cells co-transfected with pGL3-IRF3-luc/UAS-Luc and Myc-vector or Myc-YopT and poly(I:C),poly(dA:dT)respectively,it was found that compared with the control group transfected with Myc-vector,co-transfection of poly(I:C)and MycYopT decreased the activity of IRF3 luciferase,while co-transfection of poly(dA:dT)and Myc-YopT did not affect the activity of IRF3 luciferase;In HEK293T cells treated with pGL3-NF-?B-Luc and poly(I:C)or poly(dA:dT)respectively,it was found that compared with the control group transfected with Myc-vector,the activity of luciferase of NF-?B was decreased by co-transfection of poly(I:C)and Myc-YopT,while cotransfection of poly(dA:dT)and Myc-YopT did not affect the luciferase activity of NF?B.In HEK293T cells treated with pGL3-IRF3-luc/UAS-Luc and Myc-vector or MycYopT,respectively,compared with the control group transfected with Flag-vector,it was found that cotransfection of Flag-RIG-I and Myc-YopT decreased the luciferase activity of IRF3,while other groups did not affect the luciferase activity of IRF3;In HEK293T cells transfected with pGL3-NF-? B-Luc and Myc-vector or Myc-YopT respectively,it was found that compared with the control group transfected with Flag-vector,cotransfection of Flag-RIG-I and Myc-YopT decreased the luciferase activity of NF-? B,but not in other groups.Part ?:YopT-mediated degradation of RIG-I:In HEK293T cells co-transfected with Flag-RIG-I and different doses of Myc-YopT,it was found that the protein expression level of Flag-RIG-I decreased gradually with the increase of Myc-YopT transfection;In HEK293T cells co-transfected with Flag-RIG-I and Myc-vector,Myc-YopJ,Myc-YopE,Myc-YopM,Myc-YopT,it was found that overtransfection of Myc-YopT decreased the protein expression of Flag-RIG-I compared with the control group transfected with Myc-vector,while other groups did not affect the protein expression of Flag-RIG-I;After 24 hours of culture in HEK293T cells of FlagRIG-I and Myc-vector,Myc-YopT,respectively,and then treated with cycloheximide(50 mm)for different times,it was found that overtransfection of Myc-YopT shortened the half-life of Flag-RIG-I compared with the control group transfected with Myc-vector;In RAW264.7 cells transfected with siCtrl,siRIG-I-1,siRIG-I-2,siRIG-I-3 respectively,it was found that the level of RIG-I protein in other groups decreased compared with the control group transfected with siCtrl;Wild type RAW264.7 cells or siRIG-I RAW264.7 cells were infected with Yersinia pestis wild(WT)strain,defective strain(? YopT)or replicative strain(? YopT/YopT),respectively.It was found that the level of TBK1 phosphorylation and RIG-I protein expression decreased after infection with WT strain and ? YopT/YopT strain;Wild type RAW264.7 cells or RAW264.7 cells of siRIG-I were infected with wild(WT)strain,defective(? YopT)strain or replicative(? YopT/YopT)strain of Yersinia pestis,respectively.It was found that the level of IFN-? mRNA in the cells infected with WT strain and ? YopT/YopT strain was down-regulated;Wildtype RAW264.7 cells or RAW264.7 cells of siRIG-I were infected with wild(WT)strain,defective strain(? YopT)or replicative strain(? YopT/YopT),respectively.It was found that the level of IL-6 mRNA in the cells infected with WT strain and ? YopT/YopT strain was down-regulated.Part ?:YopT increases the ubiquitin level of K48-connected RIG-I:In HEK293T cells co-transfected with Flag-RIG-I-HA and Myc-vector,Myc-YopT,it was found that compared with the control group cotransfected with Myc-vector,the protein size of Flag-RIG-I-HA did not change after overtransfection of Myc-YopT,but the protein expression level decreased;In HEK293T cells co-transfected with Flag-RIGI,HA-Ub and RIG-I agonist 5'ppp-dsRNA and Myc-vector,Myc-YopT,respectively,it was found that the ubiquitin level of Flag-RIG increased after overtransfection of MycYopT compared with the control group transfected with Myc-vector;In HEK293T cells co-transfected with Flag-RIG-I,HA-K48-Ub and RIG-I agonist 5'ppp-dsRNA and Mycvector,Myc-YopT,respectively,it was found that compared with the control group cotransfected with Myc-vector,the ubiquitin level of Flag-RIG at position 48 lysine connection was increased after overtransfection of Myc-YopT;In RAW264.7 cells cotransfected with RIG-I agonist 5'ppp-dsRNA and Myc-vector,Myc-YopT,it was found that the ubiquitin level of RIG-I increased after overtransfection of Myc-YopT compared with the control group transfected with Myc-vector.Conclusion:Through the above results,we found that YopT acts on RIG-I protein to increase the K48 polymerized ubiquitin conjugate,thereby destroying the stability of RIG-I and inhibiting the activated molecules in innate immunity. |
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