鼠疫耶尔森菌外膜蛋白J参与宿主免疫逃逸机制的研究

发布时间：2018-05-13 13:52

本文选题：Y.pestis + YopJ　；参考：《吉林大学》2017年博士论文

【摘要】：鼠疫是一种人畜共患烈性传染病,一般通过跳蚤在啮齿类动物之间的传播而流行,当人接触到被感染的跳蚤或啮齿类动物时则有可能引发鼠疫。鼠疫的致病菌鼠疫耶尔森菌(Yersinia pestis,Y.pestis)可以通过三型分泌系统(Type III secretion system,T3SS)将其外膜蛋白(Yersinia outer proteins,Yops)注入宿主细胞中。其中,Yop J是Y.pestis非常重要的外膜蛋白效应因子,它具有去泛素化酶和乙酰基转移酶两种活性,既可以使转化因子生长激酶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)、肿瘤坏死因子受体相关因子2(TNF receptor associated factor 2,TRAF2)及肿瘤坏死因子受体相关因子6(TNF receptor associated factor 6,TRAF6)去泛素化,又可以通过乙酰化IκB激酶(inhibitor of nuclear factor kappa-B kinases,IKKs)和有丝分裂原活化蛋白激酶激酶(mitogen-activated protein kinase kinases,MKKs)的丝氨酸和苏氨酸活性位点,进一步抑制其磷酸化级联反应,在Y.pestis逃逸宿主细胞免疫应答过程中具有非常重要的作用。因此,研究Yop J的功能并寻找其宿主内靶标对深入了解Y.pestis的致病机制具有十分重要的意义。先天性免疫系统几乎存在于所有的多细胞动物中,是机体十分重要的抗感染机制之一。它主要通过模式识别受体(pattern recognition receptor,PRR)识别病原相关分子模式(pathogen-associated molecular pattern,PAMP),从而引起一系列的信号转导及生理生化反应,最终产生多种细胞因子、趋化因子和共刺激分子等效应因子抵抗病原体的侵染。先天性免疫系统中的干扰素刺激因子(stimulator of interferon genes,STING)既可以作为接头蛋白接收RIG-I样受体(RIG-I like receptors,RLRs)传来的信号,也可以作为一个独立的感受器识别胞质中的环单磷酸鸟苷-单磷酸腺苷合成酶(cyclic GMP-AMP synthase,c GAS)、环二鸟苷酸(cyclic diguanylate monophosphate,c-di-GMP)、病原微生物DNA和RNA。STING被激活后从内质网转移到高尔基体并与TANK结合激酶1(TANK-binding kinase 1,TBK1)结合形成复合物,辅助TBK1的磷酸化作用,进而激活干扰素调节因子3(interferon regulatory factor 3,IRF3),最终引起细胞因子的产生和释放。STING作为先天性免疫系统中非常重要的信号蛋白,可以识别多种病原体,从而介导病原体对宿主先天性免疫应答的逃逸。Y.pestis既可以通过Yop E、Yop H、Yop T和Ypk A的协同作用破坏细胞骨架、抵抗细胞的内吞,又可以通过Yop J阻断宿主的信号转导抑制先天性免疫反应,以利于病原体在宿主细胞中建立持续的感染。鉴于Yop J对Y.pestis的致病性和毒力传播的重要作用,我们通过酵母双杂交实验在先天性免疫信号通路中筛选Yop J作用的新靶点,前期研究发现Yop J有可能与干扰素刺激因子STING存在相互作用,进一步的研究表明:1.Yop J通过对STING进行63位赖氨酸(lysine,Lys)去泛素化抑制胞质DNA所引起的IRF3激活:通过双荧光素酶报告基因实验发现Y.pestis DNA或c-di-GMP对IRF3的激活可以被Yop J特异性的抑制,并呈剂量依赖性。通过功能性实验发现Yop J可以对STING进行K63位去泛素化,而Yop J C172A则没有这种作用,Yop J及Yop J C172A对STING的乙酰化均没有影响。2.Yop J通过与STING相互作用下调其稳定性并影响STING-TBK1复合物的形成:免疫荧光和免疫共沉淀实验结果表明,Yop J通过与STING相互作用影响其从内质网向高尔基体的转移,进而影响STING-TKB1复合物的形成。菌感染实验结果表明,相对于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)可以特异的下调STING的蛋白稳定性。半衰期检测实验、荧光定量PCR和免疫印迹实验结果表明,Yop J可以缩短STING的半衰期并影响其向IRF3信号的传递,但对STING m RNA的稳定性并没有影响。3.Yop J通过其第172位半胱氨酸(cysteine,Cys)作用于STING影响IRF3免疫反应对Y.pestis在宿主细胞中建立持续感染均有十分重要的辅助作用:通过双荧光素酶报告基因实验、磷酸化实验、菌感染实验和荧光定量PCR的方法发现,相对于Yop J C172A及Y.pestisΔYop J/Yop J C172A突变回复菌(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缺失回复菌(Y.pestis lacking Yop J with Yop J resue,ΔYop J/Yop J Y.pestis)对STING有明显的的去泛素化作用,并可以抑制IRF3的激活。通过菌成活率实验发现,相对于ΔYop J Y.pestis和ΔYop J/Yop J C172A Y.pestis,WT Y.pestis和ΔYop J/Yop J Y.pestis在宿主细胞内具有更高的成活率。通过小鼠感染实验发现,相对于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感染的小鼠中检测到了更低水平的STING,并且组织炎症和坏死情况也明显减少。通过上述研究,我们首次发现了Yop J靶向于宿主先天性免疫的新靶点STING,并阐述了Yop J作用于STING的分子机制及这种机制对Y.pestis在宿主细胞中建立持续感染的重要作用,揭示了Y.pestis外膜蛋白效应因子调节先天性免疫的新方式,为鼠疫的预防及治疗提供了新的思路和方向,具有十分重要的理论和临床研究意义。
[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.

【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R392

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