【摘要】：結(jié)核病仍然是全世界高致死的疾病。結(jié)核分枝桿菌是人類結(jié)核病致病菌,是迄今為止最成功的人類病原菌能存活在巨噬細胞中。結(jié)核分枝桿菌在感染中,可以感知環(huán)境細微的改變和抵抗代謝壓力,如低pH值、活性氮物種(RNS)、活性氧(ROS)和其他抗菌因子。病原體毒力因子在緩沖抗菌物質(zhì)和決定感染的結(jié)果中具有重要作用。Rv0341是一個功能未知的蛋白,并且有研究證明在人類結(jié)核肉芽腫、巨噬細胞感染和抗生素的壓力下表達上調(diào)。為了探究結(jié)核分枝桿菌Rv0341基因在抗生素耐受、致病性或毒力方面發(fā)揮的作用,我們在快生型分枝桿菌模式菌株-恥垢分枝桿菌中異源表達了Rv0341蛋白。本研究發(fā)現(xiàn)與空載菌株相比重組菌Ms_Rv0341具有顯著的表型差異,重組菌Ms_Rv0341改變了恥垢分枝桿菌的單菌落形態(tài)、滑動能力和生物膜的形成。此外,我們發(fā)現(xiàn)Rv0341增加了恥垢分枝桿菌對糖肽類抗生素萬古霉素和替考拉寧的耐受能力。而且在恥垢分枝桿菌中過表達Rv0341使恥垢分枝桿菌對低pH、SDS和氧化壓力(H2O2和聯(lián)氨)等壓力更加耐受。這些表型的產(chǎn)生與在Ms_Rv0341中增加了壓力耐受相關(guān)基因的表達有關(guān)。Rv0341蛋白提高了恥垢分枝桿菌在胞內(nèi)存活率。通過測定溴化乙錠的攝取量來檢測重組菌細胞壁的滲透性的實驗中,我們發(fā)現(xiàn)與空載菌Ms_Vec相比重組菌Ms_Rv0341的細胞壁滲透性顯著降低。這些數(shù)據(jù)初次證明了Rv0341通過改變細胞壁滲透性在分枝桿菌生理和致病機理中具有重要功能。
[Abstract]:Tuberculosis remains the leading cause of death worldwide. Mycobacterium tuberculosis is the most successful pathogen of human tuberculosis, which can survive in macrophages. Mycobacterium tuberculosis can sense minor changes in the environment and resist metabolic pressures such as low pH, active nitrogen species (RNS), reactive oxygen (ROS) and other antimicrobial factors. Pathogen virulence factors play an important role in buffering antibacterial substances and determining the results of infection. Rv0341 is an unknown protein and has been demonstrated to be up-regulated under the pressure of human tuberculosis granuloma macrophage infection and antibiotics. In order to explore the role of Rv0341 gene in antibiotic resistance, pathogenicity or virulence, we expressed Rv0341 protein in Mycobacterium smearus, a model strain of Mycobacterium spp. It was found that the recombinant strain Ms_Rv0341 had significant phenotypic difference compared with the non-loaded strain. The recombinant strain Ms_Rv0341 changed the colony morphology, sliding ability and biofilm formation of Mycobacterium smegmatis. In addition, we found that Rv0341 increased the tolerance of Mycobacterium smegmatis to glycopeptide antibiotics vancomycin and teicoplanin. Moreover, the overexpression of Rv0341 in Mycobacterium smearus made the strain more tolerant to low pH,SDS and oxidative pressure (H2O2 and hydrazine). These phenotypes were associated with increased expression of stress tolerance related genes in Ms_Rv0341. Rv0341 protein increased the intracellular survival rate of Mycobacterium smegmatis. By measuring the uptake of ethidium bromide to determine the cell wall permeability of the recombinant bacteria, we found that the cell wall permeability of the recombinant strain Ms_Rv0341 was significantly lower than that of the unloaded bacteria Ms_Vec. These data demonstrate for the first time that Rv0341 plays an important role in mycobacterium physiology and pathogenicity by changing cell wall permeability.
【學(xué)位授予單位】：西南大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R378.911

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