QS系统VjbR在布鲁氏菌胞内生存中作用的研究
发布时间：2019-02-13 14:54
【摘要】： 布鲁氏菌病是一种危害严重的人兽共患病,在世界范围内有广泛流行,给人类健康和经济发展带来巨大损失。布鲁氏菌是一种胞内寄生菌,深入探讨其独特的胞内生存机制和毒力因素,对于布鲁氏菌致病机制的理解、新型疫苗的研发以及布鲁氏菌病的临床治疗等具有重要意义。 布鲁氏菌有很多感应和处理环境信号的调控系统及毒力因子,如QS密度感应系统。当布鲁氏菌侵入宿主细胞时,QS系统可以调控布鲁氏菌各种靶基因以适应胞内各种不利的环境信号,从而增强抵抗巨噬细胞杀伤的能力。布鲁氏菌QS系统有两个调控蛋白:VjbR和BlxR。目前对BlxR的毒力表型以及调控机制已经进行了详细的研究,但是对VjbR的调控机制尚不完全清楚。另外,本实验室前期研究中发现,T4SS的virB操纵子对vjbR有正调控作用,且virB是细菌重要的毒力基因,可增强布鲁氏菌胞内存活能力。同时已有文献报道virB和vjbR关系密切,相互影响。那么vjbR是否与细菌毒力表型相关?是否可以调控virB从而影响布鲁氏菌胞内生存?vjbR的调控机制是怎样的呢?这些问题的回答,有利于解释和深入探讨布鲁氏菌胞内生存机制和致病机制。 为研究布鲁氏菌QS系统调控蛋白VjbR与布鲁氏菌毒力和胞内生存的关系,阐明其调控机制,本研究首先构建vjbR基因缺失突变株和互补株。通过对突变株、互补株和野生株生长曲线的观察,发现野生株的生长速度较突变株和互补株快,提示vjbR可能通过调控特定基因而正调控布鲁氏菌的生长。胞内存活及小鼠体内生存等表型实验显示,vjbR突变株虽然可以入侵巨噬细胞并在小鼠的脾脏和肝脏细胞内生存,但生存能力减弱,同时,我们在体外模拟了巨噬细胞内的多种胁迫条件,如高盐、高渗、酸、热休克和氧压力等,与野生株和互补株相比,突变株在这些刺激条件下的生存率都有不同程度的降低,表明vjbR对于布鲁氏菌适应胞内恶性环境、抵抗环境压力、建立慢性感染是必需的。与此同时,我们又研制了布鲁氏菌全基因组DNA芯片。利用全基因组芯片,比较分析了vjbR突变株与16M野生株转录谱差异,鉴定vjbR调控的靶基因,并对这些基因的功能进行详细阐述,为QS调控网络的研究提供靶标基因。本研究中,我们对布鲁氏菌QS系统在毒力中发挥的作用及其调控机制进行了详细的探讨,为布鲁氏菌治病机制的研究提供大量有价值的信息。
[Abstract]:Brucellosis is a serious zoonosis, which is widespread in the world and brings great loss to human health and economic development. Brucella is a kind of intracellular parasitic bacteria. It is of great significance to understand the pathogenic mechanism of Brucella, to develop new vaccine and to treat brucellosis, because of its unique intracellular survival mechanism and virulence factors. Brucella has many regulatory systems for sensing and processing environmental signals and virulence factors, such as QS density sensing systems. When Brucella invades host cells, the QS system can regulate various target genes of Brucella to adapt to various adverse environmental signals in the cell, thus enhancing the ability to resist the killing of macrophages. Brucella QS system has two regulatory proteins: VjbR and BlxR. At present, the virulence phenotype and regulatory mechanism of BlxR have been studied in detail, but the regulatory mechanism of VjbR is not completely clear. In addition, we found that the virB operon of T4SS has positive regulation on vjbR, and virB is an important virulence gene of bacteria, which can enhance the intracellular viability of Brucella. At the same time, it has been reported that virB and vjbR are closely related to each other. So is vjbR associated with bacterial virulence phenotype? Can virB be regulated to affect the intracellular survival of Brucella? what is the regulatory mechanism of vjbR? The answers to these questions are helpful to explain and explore the intracellular survival and pathogenic mechanisms of Brucella. In order to study the relationship between the QS regulatory protein VjbR and the virulence and intracellular survival of Brucella, and to elucidate its regulatory mechanism, we first constructed the mutant and complementary strain of vjbR gene deletion. By observing the growth curves of mutants, complementary plants and wild plants, it was found that the growth rate of wild plants was faster than that of mutants and complementary plants, suggesting that vjbR may be regulating the growth of Brucella by regulating specific genes. Phenotypic experiments showed that vjbR mutant could invade macrophages and survive in mouse spleen and liver cells, but its viability was weakened. We simulated a variety of stress conditions in macrophages in vitro, such as high salt, hyperosmotic, acid, heat shock, and oxygen pressure. The survival rate of mutant strains decreased in varying degrees compared with wild and complementary strains. It was suggested that vjbR was necessary for brucella to adapt to malignant intracellular environment, resist environmental stress and establish chronic infection. At the same time, we developed the whole genome DNA chip of Brucella. The transcriptional differences between vjbR mutant and 16m wild strain were compared and analyzed by using the whole genome chip. The target genes regulated by vjbR were identified, and the functions of these genes were described in detail, which provided target genes for the study of QS regulatory network. In this study, we discussed in detail the role of brucella QS system in virulence and its regulatory mechanism, and provided a lot of valuable information for the study of brucellosis treatment mechanism.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2009
【分类号】：R378.51
本文编号：2421666
[Abstract]:Brucellosis is a serious zoonosis, which is widespread in the world and brings great loss to human health and economic development. Brucella is a kind of intracellular parasitic bacteria. It is of great significance to understand the pathogenic mechanism of Brucella, to develop new vaccine and to treat brucellosis, because of its unique intracellular survival mechanism and virulence factors. Brucella has many regulatory systems for sensing and processing environmental signals and virulence factors, such as QS density sensing systems. When Brucella invades host cells, the QS system can regulate various target genes of Brucella to adapt to various adverse environmental signals in the cell, thus enhancing the ability to resist the killing of macrophages. Brucella QS system has two regulatory proteins: VjbR and BlxR. At present, the virulence phenotype and regulatory mechanism of BlxR have been studied in detail, but the regulatory mechanism of VjbR is not completely clear. In addition, we found that the virB operon of T4SS has positive regulation on vjbR, and virB is an important virulence gene of bacteria, which can enhance the intracellular viability of Brucella. At the same time, it has been reported that virB and vjbR are closely related to each other. So is vjbR associated with bacterial virulence phenotype? Can virB be regulated to affect the intracellular survival of Brucella? what is the regulatory mechanism of vjbR? The answers to these questions are helpful to explain and explore the intracellular survival and pathogenic mechanisms of Brucella. In order to study the relationship between the QS regulatory protein VjbR and the virulence and intracellular survival of Brucella, and to elucidate its regulatory mechanism, we first constructed the mutant and complementary strain of vjbR gene deletion. By observing the growth curves of mutants, complementary plants and wild plants, it was found that the growth rate of wild plants was faster than that of mutants and complementary plants, suggesting that vjbR may be regulating the growth of Brucella by regulating specific genes. Phenotypic experiments showed that vjbR mutant could invade macrophages and survive in mouse spleen and liver cells, but its viability was weakened. We simulated a variety of stress conditions in macrophages in vitro, such as high salt, hyperosmotic, acid, heat shock, and oxygen pressure. The survival rate of mutant strains decreased in varying degrees compared with wild and complementary strains. It was suggested that vjbR was necessary for brucella to adapt to malignant intracellular environment, resist environmental stress and establish chronic infection. At the same time, we developed the whole genome DNA chip of Brucella. The transcriptional differences between vjbR mutant and 16m wild strain were compared and analyzed by using the whole genome chip. The target genes regulated by vjbR were identified, and the functions of these genes were described in detail, which provided target genes for the study of QS regulatory network. In this study, we discussed in detail the role of brucella QS system in virulence and its regulatory mechanism, and provided a lot of valuable information for the study of brucellosis treatment mechanism.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2009
【分类号】：R378.51
【引证文献】
相关硕士学位论文 前1条
1 王同坤;布鲁氏菌非编码小RNA的预测、鉴定及BSR-16的功能分析[D];吉林大学;2011年
，本文编号：2421666
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