The Research On The Mechanism Of Yersinia Outer Protein J Participating In Immunologic Escape Of The Host

Plague is an acute and highly infectious disease,which is generally prevalent through the spread of fleas among rodents.When people are exposed to the infected fleas or rodents,there will be epidemics of plague in humans.Yersinia pestis(Y.pestis),pathogen of plague,has acquired strategies to escape host immune system by injecting effector proteins into host cells by using type III secretion system(T3SS).Y.pestis injects at least 6 kinds of Yersinia outer proteins(Yops)into the infected host cells,resulting in the inhibition of the inherent immune signaling,which ultimately allows the bacteria to establish a systemic infection.Through inhibiting the innate immune response,Yop J nullifies the activation of pro-inflammatory cytokines,and subsequently induces macrophage apoptosis to contribute to successful infection.The host innate immune system provides the first line of defense against infectious agents,including pathogenic bacteria.Under physiological conditions,most bacterial colonization in host cells can be restrained by innate immune system.When pathogenic bacteria invade hosts,the pathogen associated molecule patterns(PAMPs)can be recognized by pattern recognition receptors(PRRs)and then trigger the rapid induction of cytokines to eliminate the pathogens.Several independent groups have identified stimulator of interferon genes(STING)as a central and multifaceted mediator of the innate immune response.Later studies show that STING acts as not only an adaptor,but also a direct sensor of cytosolic DNA and cyclic diguanylate monophosphate(c-di-GMP).Cytosolic detection of pathogen-derived DNA and c-di-GMP are the major mechanisms of bacterial-induced interferon production.STING is ubiquitinated during the invasion of certain nucleic acids and then separated from the endoplasmic reticulum(ER)to form complexes with TBK1 at the Golgi apparatus.The STING-TBK1 complexes further activate the IRF3,and the phosphorylated IRF3 forms dimers to activate transcription of downstream genes inthe nucleus.Therefore,STING functions as both an adaptor and a sensor,and has been demonstrated to facilitate downstream signal transmission to IRF3 and nuclear factor ?B(NF-?B).Whereas Yop J is important for Y.pestis,the potential new targets of Yop J are under investigation.Via the yeast two-hybrid experiment,we found Yop J might interact with STING.Further experiments showed that:1.Yop J reduces K63-linked ubiquitination signals of STING and inhibits cytosolic DNA antibacterial signaling: Dual luciferase reporter assays indicated that IRF3 could be stimulated by both the genomic DNA of Y.pestis and c-di-GMP and the activation of IRF3 could be inhibited by Yop J in a dose dependent manner.Acetylization and ubiquitination assays showed that Yop J but not Yop J C172 A reduced K63-linked ubiquitination signals of STING,while neither Yop J nor Yop J C172 A had effect on acetylization of STING.2.Yop J blocks IRF3 signaling by inducing STING for degradation:Immunofluorescence and co-immunoprecipitation indicated that Yop J interacted and prevented STING from transfering from ER to Golgi apparatus and affected the interaction between STING and TKB1.Bacterial infection assays indicated that the wild type Y.pestis(WT Y.pestis)could downregulate its stability comparing with that of Y.pestis lacking Yop J(?Yop J Y.pestis).Cycloheximide chase assays,q PCR and WB indicated that the STING expression was considerably reduced by Yop J introduction,but there no change in endogenous STING m RNA level with increased Yop J expression.3.Yop J 172 th cysteine is the active site for blocking STING-mediated signaling and the function of Yop J contributes to bacterial virulence: Dual luciferase reporter assays indicated that the levels both of STING protein abundance and IRF3 phosphorylation were diminished considerably by WT Yop J,but not Yop J C172 A.Bacterial infection assays showed that the mean numbers of bacteria recovered from WT and ?Yop J/Yop J Y.pestis-infected BMDMs were considerably higher than those from ?Yop J and ?Yop J/Yop J C172 A Y.pestis-infected BMDMs.Mice infectionassays showed that a significantly increased inflammation,tissue injury,necrosis and decreased staining of STING in ?Yop J or ?Yop J/Yop J C172 A bacteria infected tissues,while WT and ?Yop J/Yop J strains generally caused lower levels of inflammation and necrosis.Together,we show that Yop J can inhibit the activation of IFN-? production in macrophages by binding to STING and removing its K63-linked ubiquitination.Those results not only help us to understand the mechanism of pestis to evade the host immunity,but also suggest that Yop J may form a possible target for pestis treatment.