本文選題：Y.pestis + YopJ�。� 參考：《吉林大學(xué)》2017年博士論文

【摘要】：鼠疫是一種人畜共患烈性傳染病,一般通過跳蚤在嚙齒類動物之間的傳播而流行,當(dāng)人接觸到被感染的跳蚤或嚙齒類動物時則有可能引發(fā)鼠疫。鼠疫的致病菌鼠疫耶爾森菌(Yersinia pestis,Y.pestis)可以通過三型分泌系統(tǒng)(Type III secretion system,T3SS)將其外膜蛋白(Yersinia outer proteins,Yops)注入宿主細胞中。其中,Yop J是Y.pestis非常重要的外膜蛋白效應(yīng)因子,它具有去泛素化酶和乙酰基轉(zhuǎn)移酶兩種活性,既可以使轉(zhuǎn)化因子生長激酶1(TGF-βactivated kinase 1,TAK-1)、NF-κB抑制蛋白(inhibitor of nuclear factor kappa-B,IκB)、IκB激酶β(inhibitor of nuclear factor kappa-B kinaseβ,IKKβ)、腫瘤壞死因子受體相關(guān)因子2(TNF receptor associated factor 2,TRAF2)及腫瘤壞死因子受體相關(guān)因子6(TNF receptor associated factor 6,TRAF6)去泛素化,又可以通過乙�；疘κB激酶(inhibitor of nuclear factor kappa-B kinases,IKKs)和有絲分裂原活化蛋白激酶激酶(mitogen-activated protein kinase kinases,MKKs)的絲氨酸和蘇氨酸活性位點,進一步抑制其磷酸化級聯(lián)反應(yīng),在Y.pestis逃逸宿主細胞免疫應(yīng)答過程中具有非常重要的作用。因此,研究Yop J的功能并尋找其宿主內(nèi)靶標(biāo)對深入了解Y.pestis的致病機制具有十分重要的意義。先天性免疫系統(tǒng)幾乎存在于所有的多細胞動物中,是機體十分重要的抗感染機制之一。它主要通過模式識別受體(pattern recognition receptor,PRR)識別病原相關(guān)分子模式(pathogen-associated molecular pattern,PAMP),從而引起一系列的信號轉(zhuǎn)導(dǎo)及生理生化反應(yīng),最終產(chǎn)生多種細胞因子、趨化因子和共刺激分子等效應(yīng)因子抵抗病原體的侵染。先天性免疫系統(tǒng)中的干擾素刺激因子(stimulator of interferon genes,STING)既可以作為接頭蛋白接收RIG-I樣受體(RIG-I like receptors,RLRs)傳來的信號,也可以作為一個獨立的感受器識別胞質(zhì)中的環(huán)單磷酸鳥苷-單磷酸腺苷合成酶(cyclic GMP-AMP synthase,c GAS)、環(huán)二鳥苷酸(cyclic diguanylate monophosphate,c-di-GMP)、病原微生物DNA和RNA。STING被激活后從內(nèi)質(zhì)網(wǎng)轉(zhuǎn)移到高爾基體并與TANK結(jié)合激酶1(TANK-binding kinase 1,TBK1)結(jié)合形成復(fù)合物,輔助TBK1的磷酸化作用,進而激活干擾素調(diào)節(jié)因子3(interferon regulatory factor 3,IRF3),最終引起細胞因子的產(chǎn)生和釋放。STING作為先天性免疫系統(tǒng)中非常重要的信號蛋白,可以識別多種病原體,從而介導(dǎo)病原體對宿主先天性免疫應(yīng)答的逃逸。Y.pestis既可以通過Yop E、Yop H、Yop T和Ypk A的協(xié)同作用破壞細胞骨架、抵抗細胞的內(nèi)吞,又可以通過Yop J阻斷宿主的信號轉(zhuǎn)導(dǎo)抑制先天性免疫反應(yīng),以利于病原體在宿主細胞中建立持續(xù)的感染。鑒于Yop J對Y.pestis的致病性和毒力傳播的重要作用,我們通過酵母雙雜交實驗在先天性免疫信號通路中篩選Yop J作用的新靶點,前期研究發(fā)現(xiàn)Yop J有可能與干擾素刺激因子STING存在相互作用,進一步的研究表明:1.Yop J通過對STING進行63位賴氨酸(lysine,Lys)去泛素化抑制胞質(zhì)DNA所引起的IRF3激活:通過雙熒光素酶報告基因?qū)嶒灠l(fā)現(xiàn)Y.pestis DNA或c-di-GMP對IRF3的激活可以被Yop J特異性的抑制,并呈劑量依賴性。通過功能性實驗發(fā)現(xiàn)Yop J可以對STING進行K63位去泛素化,而Yop J C172A則沒有這種作用,Yop J及Yop J C172A對STING的乙�；鶝]有影響。2.Yop J通過與STING相互作用下調(diào)其穩(wěn)定性并影響STING-TBK1復(fù)合物的形成:免疫熒光和免疫共沉淀實驗結(jié)果表明,Yop J通過與STING相互作用影響其從內(nèi)質(zhì)網(wǎng)向高爾基體的轉(zhuǎn)移,進而影響STING-TKB1復(fù)合物的形成。菌感染實驗結(jié)果表明,相對于Y.pestis Yop J敲除菌(Y.pestis lacking Yop J,ΔYop J Y.pestis),野生型Y.pestis(wild type Yersinia pestis,WT Y.pestis)可以特異的下調(diào)STING的蛋白穩(wěn)定性。半衰期檢測實驗、熒光定量PCR和免疫印跡實驗結(jié)果表明,Yop J可以縮短STING的半衰期并影響其向IRF3信號的傳遞,但對STING m RNA的穩(wěn)定性并沒有影響。3.Yop J通過其第172位半胱氨酸(cysteine,Cys)作用于STING影響IRF3免疫反應(yīng)對Y.pestis在宿主細胞中建立持續(xù)感染均有十分重要的輔助作用:通過雙熒光素酶報告基因?qū)嶒�、磷酸化實驗、菌感染實驗和熒光定量PCR的方法發(fā)現(xiàn),相對于Yop J C172A及Y.pestisΔYop J/Yop J C172A突變回復(fù)菌(Y.pestis lacking Yop J with Yop J C172A resue,ΔYop J/Yop J C172A Y.pestis),Yop J和Y.pestisΔYop J/Yop J缺失回復(fù)菌(Y.pestis lacking Yop J with Yop J resue,ΔYop J/Yop J Y.pestis)對STING有明顯的的去泛素化作用,并可以抑制IRF3的激活。通過菌成活率實驗發(fā)現(xiàn),相對于ΔYop J Y.pestis和ΔYop J/Yop J C172A Y.pestis,WT Y.pestis和ΔYop J/Yop J Y.pestis在宿主細胞內(nèi)具有更高的成活率。通過小鼠感染實驗發(fā)現(xiàn),相對于ΔYop J Y.pestis和ΔYop J/Yop J C172A Y.pestis感染的小鼠,在WT Y.pestis和ΔYop J/Yop J Y.pestis感染的小鼠中檢測到了更低水平的STING,并且組織炎癥和壞死情況也明顯減少。通過上述研究,我們首次發(fā)現(xiàn)了Yop J靶向于宿主先天性免疫的新靶點STING,并闡述了Yop J作用于STING的分子機制及這種機制對Y.pestis在宿主細胞中建立持續(xù)感染的重要作用,揭示了Y.pestis外膜蛋白效應(yīng)因子調(diào)節(jié)先天性免疫的新方式,為鼠疫的預(yù)防及治療提供了新的思路和方向,具有十分重要的理論和臨床研究意義。
[Abstract]:Plague is a kind of zoonotic infectious disease, usually through the spread of fleas in rodents, and when people come into contact with infected fleas or rodents, it may cause plague. The pathogenic bacteria of the plague (Yersinia pestis, Y.pestis) can pass the three type secretory system (Type III secretion syst). Em, T3SS) inject its outer membrane protein (Yersinia outer proteins, Yops) into the host cells. Among them, Yop J is a very important outer membrane protein effect factor of Y.pestis. It has two activities, deubiquitinase and acetyltransferase, which can make the transforming factor growth kinase 1 (TGF- beta activated kinase 1). F nuclear factor kappa-B, I kappa B), I kappa B kinase beta (inhibitor of nuclear factor), tumor necrosis factor receptor related factor (2) and tumor necrosis factor receptor related factor 6 (6) The serine and threonine active sites of the enzyme (inhibitor of nuclear factor kappa-B kinases, IKKs) and the mitogen activated protein kinase kinase (mitogen-activated protein kinase kinases, MKKs) further inhibit its phosphorylation cascade reaction, which plays a very important role in the immune response of escaping host cells. Therefore, it is of great significance to study the function of Yop J and to find its host target for understanding the pathogenesis of Y.pestis. The innate immune system is almost in all multicellular animals and is one of the most important anti infection mechanisms of the body. It is mainly through the pattern recognition receptor (pattern recognition receptor, PRR). Identification of pathogen-associated molecular pattern (PAMP), resulting in a series of signal transduction and physiological and biochemical reactions, resulting in a variety of cytokines, chemokines and costimulatory factors to resist pathogens infection. The interferon stimulating factor (stimulator of in) in the innate immune system (stimulator of in) Terferon genes, STING) can be used as a joint protein to receive signals from RIG-I like receptor (RIG-I like receptors, RLRs), and can also be used as an independent receptor to identify the cyclic guanosine monophosphate monophosphate synthetase (cyclic GMP-AMP synthase, c) in cytoplasm. P), the pathogenic microorganism DNA and RNA.STING are activated from the endoplasmic reticulum to the Golgi body and combined with the TANK binding kinase 1 (TANK-binding kinase 1, TBK1) to form a complex, assisting the phosphorylation of TBK1, and then activating the interferon regulatory factor 3 (interferon regulatory factor 3, IRF3), and eventually causing the production and release of cytokines. As a very important signal protein in the innate immune system, G can identify various pathogens and mediate the escape.Y.pestis of the pathogen on the host's congenital immune response, which can not only destroy the cytoskeleton through the synergistic action of Yop E, Yop H, Yop T and Ypk A, but also resist the endocytosis of the cell, and block the signal transduction of the host by Yop J. In view of the importance of Yop J to the pathogenicity and virulence transmission of Y.pestis, we screened the new target of Yop J by yeast two hybrid experiment in the congenital immune signal pathway. Earlier studies found that Yop J was likely to be stimulated by interferon stimulation. Factor STING interactions exist, and further studies show that 1.Yop J is activated by STING 63 bits lysine (lysine, Lys) to inhibit the activation of IRF3 in the cytoplasmic DNA: the activation of Y.pestis DNA or c-di-GMP on IRF3 is inhibited by the double luciferase reporter gene experiment, and it is dose-dependent. Through functional experiments, it is found that Yop J can make K63 deubiquitination of STING, while Yop J C172A has no such effect. Yop J and Yop J C172A have no effect on the acetylation of these compounds. Yop J affects the transfer from the endoplasmic reticulum to the Golgi body through the interaction with STING, and then affects the formation of the STING-TKB1 complex. The results of the bacterial infection experiment show that compared to the Y.pestis Yop J knockout bacteria (Y.pestis lacking Yop J, Delta Yop) can be specifically downregulated. The protein stability of TING. Half life test experiments, fluorescence quantitative PCR and immunoblotting experiments show that Yop J can shorten the half life of STING and affect the transmission of IRF3 signal, but the stability of STING m RNA does not affect the.3.Yop J through its 172nd bit cysteine. .pestis has a very important auxiliary role in the establishment of persistent infection in host cells: through the experiment of double luciferase reporter gene, phosphorylation experiment, bacterial infection experiment and fluorescence quantitative PCR method, it is found that it is relative to Yop J C172A and Y.pestis Delta Yop J/Yop J C172A mutant. J/Yop J C172A Y.pestis), Yop J and Y.pestis Delta Yop J/Yop J deletion bacteria (Y.pestis lacking) have obvious deletions and can inhibit the activation. STIs and delta Yop J/Yop J Y.pestis have higher survival rates in the host cells. Mice infected with delta Yop J Y.pestis and delta Yop J/Yop J C172A were found to be lower levels in the mice infected with delta Yop J Y.pestis and delta Yop J/Yop, and to organize inflammation and necrosis. In this study, we first discovered the new target of Yop J targeting the host's congenital immune system, STING, and described the molecular mechanism of Yop J acting on STING and the important role of this mechanism for the establishment of persistent infection in the host cells by this mechanism, and revealed that Y.pestis outer membrane protein effect factor regulates innate immunity. The new way provides a new way of thinking and direction for the prevention and treatment of plague, which is of great theoretical and clinical significance.

【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：R392

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