结核分枝杆菌蛋白PPE25在胁迫应答和免疫中的功能研究

发布时间：2018-03-17 03:11

本文选题：结核分枝杆菌　切入点：PPE家族　出处：《西南大学》2017年硕士论文　论文类型：学位论文

【摘要】：结核病由病原体结核分枝杆菌(Mycobacterium tuberculosis,Mtb)感染引起,仍然是威胁全球人类健康的重大公共卫生问题。世界上大约三分之一的人口潜伏感染Mtb,每年大约有200万人死于结核病。Mtb作为一种成功地胞内致病菌,在与其宿主长期共同演化中,形成了多种存活策略,可以调节和逃避宿主免疫应答并在宿主中持续较长时间,但是其致病机制目前仍然还不清楚。尽管目前抗结核病的药物以及新型疫苗的不断出现,但是由于抗生素的不合理使用,导致多耐药菌株、广泛耐药菌株,甚至全耐药菌株的出现以及结核病与HIV共感染,使得结核病疫情进一步恶化。因此,深入了解结核分枝杆菌的生物学功能,寻找新的药物靶标和开发新型抗结核药物对控制结核病疫情是刻不容缓的。结核分枝杆菌H37Rv基因组的一个重要标志是含有两个多基因的蛋白家族,包括PE家族(99个编码基因)和PPE家族(69个编码基因),该蛋白家族占据结核分枝杆菌基因组编码能力的10%。PE/PPE家族蛋白只存在致病性的分枝杆菌,可能是毒力因子,其功能可能与结核分枝杆菌的抗原提呈、抗原变异,调节巨噬细胞免疫效应功能相关。据报道,在结核菌感染宿主细胞过程中,大多数PPE家族蛋白上调表达并且分泌定位在细胞表面,在结核分枝杆菌与宿主间相互作用中具有重要作用。目前仅仅只有少数PPE家族蛋白被进行了深入的研究,该家族中大多数成员的功能未知。到目前为止,关于PPE25蛋白功能的研究报道很少。鸟分枝杆菌基因MAV2928(与PPE25有52%同源性)转座子插入突变,发现突变株在小鼠巨噬细胞内复制的能力减弱并且该蛋白可以阻止吞噬体/溶酶体融合。另外,MAV2928可以与MAV2921(ESAT6家族蛋白,ESX-N)相互作用,另外,在感染巨噬细胞和小鼠模型中,△PPE25-PE19缺失的结核菌株减弱胞内存活,这些结果表明PPE25可能在分枝杆菌与宿主相互作用中具有重要作用。为了研究PPE25是否在应对压力环境以及病原菌与宿主互作中发挥作用,我们以耻垢分枝杆菌为模式菌株,用穿梭质粒pNIT构建过表达Rv1787基因的重组耻垢分枝杆菌Ms_Rv1787。通过亚细胞定位实验,我们发现Rv1787蛋白定位于耻垢分枝杆菌的细胞表面。过表达Rv1787蛋白可以改变耻垢分枝杆菌菌体、菌落形态以及滑动能力。在体外各种压力条件下,过表达Rv1787蛋白增强重组菌Ms_Rv1787在氧化压力(H2O2,diamide)条件下的存活。在侵染THP-1巨噬细胞过程中,表达Rv1787蛋白的重组耻垢分枝杆菌Ms_Rv1787可以增强在巨噬细胞中的胞内存活,并且诱导低水平的促炎症细胞因子的表达,有利于自生的存活,其中suppressors of cytokine signaling3蛋白上调表达,有可能参与抑制促炎症细胞因子的表达。此外,过表达Rv1787蛋白的重组耻垢分枝杆菌Ms_Rv1787可以促进巨噬细胞中的凋亡。综上所述,我们的研究表明,PPE家族蛋白Rv1787是一个细胞被膜相关蛋白并暴露在细胞壁上,过表达Rv1787蛋白可以增强耻垢分枝杆菌对氧化压力环境的耐受,过表达Rv1787蛋白可以增强耻垢分枝杆菌在巨噬细胞中的胞内存活,通过上调suppressors of cytokine signaling3蛋白的表达,诱导低水平的促炎症细胞因子的表达,有利于自生的存活。此外,过表达Rv1787蛋白的重组耻垢分枝杆菌Ms_Rv1787可以促进巨噬细胞中的凋亡。因此,本文的研究可能会提供关于PPE家族蛋白Rv1787如何与宿主细胞相互作用的新线索。
[Abstract]:Tuberculosis by pathogen Mycobacterium tuberculosis (Mycobacterium tuberculosis, Mtb) caused by infection, is still a major public health problem of global threats to human health. About 1/3 of the world's population of Mtb latent infection, about 2 million people died of tuberculosis.Mtb as a successful intracellular pathogen and its host every year, in long-term co evolution. The formation of a variety of survival strategies, can regulate and evade the host immune response and continue for a long time in the host, but its pathogenesis is still unclear. Although the drug against tuberculosis and new vaccines continue to appear, but because of the irrational use of antibiotics leads to multiple drug resistant strains, extensive drug resistant strains, and even the whole drug resistant strains and tuberculosis and HIV co infection, the TB epidemic worsened. Therefore, in-depth understanding of Mycobacterium tuberculosis. Biological function, looking for new drug targets and the development of new anti tuberculosis drugs is urgent to control tuberculosis. An important sign of Mycobacterium tuberculosis H37Rv genome contains two genes including PE protein family, family (99 genes encoding PPE family (69) and a gene encoding the protein family). Occupation ability of Mycobacterium tuberculosis genome encoding 10%.PE/PPE family proteins exist only pathogenic mycobacteria, may be a virulence factor, its function may be related to Mycobacterium tuberculosis antigens, antigenic variation, regulation of immune effector cells, macrophage function. According to reports, the tuberculosis infection of host cells, most of the PPE family proteins were up-regulated the expression and secretion of localization on the cell surface of Mycobacterium tuberculosis and host interactions play an important role. At present, only a few PPE family proteins are In-depth study of unknown function most members of the family. So far, reports about the function of PPE25 protein of Mycobacterium avium. Few gene MAV2928 (52% homology with PPE25) transposon insertion mutation, the mutant strain was found to replicate in macrophages and weakening the ability of the protein can prevent phagosome / lysosome fusion. In addition, MAV2928 and MAV2921 (ESAT6 family protein, ESX-N) interaction, in addition, in infected macrophages and in mouse model of Mycobacterium tuberculosis Delta PPE25-PE19 deletion reduced intracellular survival, these results suggest that PPE25 may play an important role in mycobacterial host interactions. In order to study whether PPE25 play a role in response to the pressure of environment and the pathogen and host interaction, we in Mycobacterium smegmatis strain model was used. The shuttle plasmid pNIT was constructed over expression of Rv1787 gene Because the recombinant Mycobacterium smegmatis Ms_Rv1787. by subcellular localization experiment, we found that Rv1787 protein was localized in Mycobacterium smegmatis cells. Overexpression of Rv1787 can change the Mycobacterium smegmatis cell, colony morphology and sliding ability. In vitro under various stress conditions, the overexpression of Rv1787 protein in recombinant Ms_Rv1787 enhanced oxidative stress (H2O2, diamide) survival conditions. In the process of THP-1 infection of macrophages, the expression of Rv1787 protein of recombinant Mycobacterium smegmatis Ms_Rv1787 can enhance the intracellular survival in macrophages, and induced low levels of proinflammatory cytokine expression, is conducive to self survival, of which suppressors of cytokine signaling3 protein expression. May be involved in inhibiting the expression of proinflammatory cytokines. Moreover, overexpression of Rv1787 protein in recombinant Mycobacterium smegmatis can promote Ms_Rv1787 In apoptosis in macrophages. In summary, our study shows that PPE family protein Rv1787 is a cell membrane associated protein and exposed on the cell wall, the overexpression of Rv1787 protein can enhance the Mycobacterium smegmatis environment on oxidative stress tolerance, overexpression of Rv1787 can enhance Mycobacterium smegmatis in macrophage cells in survival, through the upregulation of suppressors expression of cytokine signaling3 protein, induced by low levels of proinflammatory cytokine expression, is conducive to self survival. In addition, over expression of recombinant M.smegmatis Rv1787 protein branch coli Ms_Rv1787 can induce apoptosis in macrophages. Therefore, this study may shed light on the PPE family of proteins Rv1787 how to interact with host cells of the cable.

【学位授予单位】：西南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R378.911

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