结核分枝杆菌Rv0341通过改变细胞壁的通透性在细菌生理和耐药性上发挥作用
发布时间：2018-10-15 15:32
【摘要】：结核病仍然是全世界高致死的疾病。结核分枝杆菌是人类结核病致病菌,是迄今为止最成功的人类病原菌能存活在巨噬细胞中。结核分枝杆菌在感染中,可以感知环境细微的改变和抵抗代谢压力,如低pH值、活性氮物种(RNS)、活性氧(ROS)和其他抗菌因子。病原体毒力因子在缓冲抗菌物质和决定感染的结果中具有重要作用。Rv0341是一个功能未知的蛋白,并且有研究证明在人类结核肉芽肿、巨噬细胞感染和抗生素的压力下表达上调。为了探究结核分枝杆菌Rv0341基因在抗生素耐受、致病性或毒力方面发挥的作用,我们在快生型分枝杆菌模式菌株-耻垢分枝杆菌中异源表达了Rv0341蛋白。本研究发现与空载菌株相比重组菌Ms_Rv0341具有显著的表型差异,重组菌Ms_Rv0341改变了耻垢分枝杆菌的单菌落形态、滑动能力和生物膜的形成。此外,我们发现Rv0341增加了耻垢分枝杆菌对糖肽类抗生素万古霉素和替考拉宁的耐受能力。而且在耻垢分枝杆菌中过表达Rv0341使耻垢分枝杆菌对低pH、SDS和氧化压力(H2O2和联氨)等压力更加耐受。这些表型的产生与在Ms_Rv0341中增加了压力耐受相关基因的表达有关。Rv0341蛋白提高了耻垢分枝杆菌在胞内存活率。通过测定溴化乙锭的摄取量来检测重组菌细胞壁的渗透性的实验中,我们发现与空载菌Ms_Vec相比重组菌Ms_Rv0341的细胞壁渗透性显著降低。这些数据初次证明了Rv0341通过改变细胞壁渗透性在分枝杆菌生理和致病机理中具有重要功能。
[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.
【学位授予单位】：西南大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R378.911
[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.
【学位授予单位】：西南大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R378.911
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