本文選題：結(jié)核分枝桿菌　切入點(diǎn)：PPE家族　出處：《西南大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：結(jié)核病由病原體結(jié)核分枝桿菌(Mycobacterium tuberculosis,Mtb)感染引起,仍然是威脅全球人類健康的重大公共衛(wèi)生問(wèn)題。世界上大約三分之一的人口潛伏感染Mtb,每年大約有200萬(wàn)人死于結(jié)核病。Mtb作為一種成功地胞內(nèi)致病菌,在與其宿主長(zhǎng)期共同演化中,形成了多種存活策略,可以調(diào)節(jié)和逃避宿主免疫應(yīng)答并在宿主中持續(xù)較長(zhǎng)時(shí)間,但是其致病機(jī)制目前仍然還不清楚。盡管目前抗結(jié)核病的藥物以及新型疫苗的不斷出現(xiàn),但是由于抗生素的不合理使用,導(dǎo)致多耐藥菌株、廣泛耐藥菌株,甚至全耐藥菌株的出現(xiàn)以及結(jié)核病與HIV共感染,使得結(jié)核病疫情進(jìn)一步惡化。因此,深入了解結(jié)核分枝桿菌的生物學(xué)功能,尋找新的藥物靶標(biāo)和開發(fā)新型抗結(jié)核藥物對(duì)控制結(jié)核病疫情是刻不容緩的。結(jié)核分枝桿菌H37Rv基因組的一個(gè)重要標(biāo)志是含有兩個(gè)多基因的蛋白家族,包括PE家族(99個(gè)編碼基因)和PPE家族(69個(gè)編碼基因),該蛋白家族占據(jù)結(jié)核分枝桿菌基因組編碼能力的10%。PE/PPE家族蛋白只存在致病性的分枝桿菌,可能是毒力因子,其功能可能與結(jié)核分枝桿菌的抗原提呈、抗原變異,調(diào)節(jié)巨噬細(xì)胞免疫效應(yīng)功能相關(guān)。據(jù)報(bào)道,在結(jié)核菌感染宿主細(xì)胞過(guò)程中,大多數(shù)PPE家族蛋白上調(diào)表達(dá)并且分泌定位在細(xì)胞表面,在結(jié)核分枝桿菌與宿主間相互作用中具有重要作用。目前僅僅只有少數(shù)PPE家族蛋白被進(jìn)行了深入的研究,該家族中大多數(shù)成員的功能未知。到目前為止,關(guān)于PPE25蛋白功能的研究報(bào)道很少。鳥分枝桿菌基因MAV2928(與PPE25有52%同源性)轉(zhuǎn)座子插入突變,發(fā)現(xiàn)突變株在小鼠巨噬細(xì)胞內(nèi)復(fù)制的能力減弱并且該蛋白可以阻止吞噬體/溶酶體融合。另外,MAV2928可以與MAV2921(ESAT6家族蛋白,ESX-N)相互作用,另外,在感染巨噬細(xì)胞和小鼠模型中,△PPE25-PE19缺失的結(jié)核菌株減弱胞內(nèi)存活,這些結(jié)果表明PPE25可能在分枝桿菌與宿主相互作用中具有重要作用。為了研究PPE25是否在應(yīng)對(duì)壓力環(huán)境以及病原菌與宿主互作中發(fā)揮作用,我們以恥垢分枝桿菌為模式菌株,用穿梭質(zhì)粒pNIT構(gòu)建過(guò)表達(dá)Rv1787基因的重組恥垢分枝桿菌Ms_Rv1787。通過(guò)亞細(xì)胞定位實(shí)驗(yàn),我們發(fā)現(xiàn)Rv1787蛋白定位于恥垢分枝桿菌的細(xì)胞表面。過(guò)表達(dá)Rv1787蛋白可以改變恥垢分枝桿菌菌體、菌落形態(tài)以及滑動(dòng)能力。在體外各種壓力條件下,過(guò)表達(dá)Rv1787蛋白增強(qiáng)重組菌Ms_Rv1787在氧化壓力(H2O2,diamide)條件下的存活。在侵染THP-1巨噬細(xì)胞過(guò)程中,表達(dá)Rv1787蛋白的重組恥垢分枝桿菌Ms_Rv1787可以增強(qiáng)在巨噬細(xì)胞中的胞內(nèi)存活,并且誘導(dǎo)低水平的促炎癥細(xì)胞因子的表達(dá),有利于自生的存活,其中suppressors of cytokine signaling3蛋白上調(diào)表達(dá),有可能參與抑制促炎癥細(xì)胞因子的表達(dá)。此外,過(guò)表達(dá)Rv1787蛋白的重組恥垢分枝桿菌Ms_Rv1787可以促進(jìn)巨噬細(xì)胞中的凋亡。綜上所述,我們的研究表明,PPE家族蛋白R(shí)v1787是一個(gè)細(xì)胞被膜相關(guān)蛋白并暴露在細(xì)胞壁上,過(guò)表達(dá)Rv1787蛋白可以增強(qiáng)恥垢分枝桿菌對(duì)氧化壓力環(huán)境的耐受,過(guò)表達(dá)Rv1787蛋白可以增強(qiáng)恥垢分枝桿菌在巨噬細(xì)胞中的胞內(nèi)存活,通過(guò)上調(diào)suppressors of cytokine signaling3蛋白的表達(dá),誘導(dǎo)低水平的促炎癥細(xì)胞因子的表達(dá),有利于自生的存活。此外,過(guò)表達(dá)Rv1787蛋白的重組恥垢分枝桿菌Ms_Rv1787可以促進(jìn)巨噬細(xì)胞中的凋亡。因此,本文的研究可能會(huì)提供關(guān)于PPE家族蛋白R(shí)v1787如何與宿主細(xì)胞相互作用的新線索。
[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.

【學(xué)位授予單位】：西南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R378.911

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本文編號(hào)：1622886