QS系統(tǒng)VjbR在布魯氏菌胞內(nèi)生存中作用的研究
發(fā)布時間：2019-02-13 14:54
【摘要】： 布魯氏菌病是一種危害嚴重的人獸共患病,在世界范圍內(nèi)有廣泛流行,給人類健康和經(jīng)濟發(fā)展帶來巨大損失。布魯氏菌是一種胞內(nèi)寄生菌,深入探討其獨特的胞內(nèi)生存機制和毒力因素,對于布魯氏菌致病機制的理解、新型疫苗的研發(fā)以及布魯氏菌病的臨床治療等具有重要意義。 布魯氏菌有很多感應和處理環(huán)境信號的調(diào)控系統(tǒng)及毒力因子,如QS密度感應系統(tǒng)。當布魯氏菌侵入宿主細胞時,QS系統(tǒng)可以調(diào)控布魯氏菌各種靶基因以適應胞內(nèi)各種不利的環(huán)境信號,從而增強抵抗巨噬細胞殺傷的能力。布魯氏菌QS系統(tǒng)有兩個調(diào)控蛋白:VjbR和BlxR。目前對BlxR的毒力表型以及調(diào)控機制已經(jīng)進行了詳細的研究,但是對VjbR的調(diào)控機制尚不完全清楚。另外,本實驗室前期研究中發(fā)現(xiàn),T4SS的virB操縱子對vjbR有正調(diào)控作用,且virB是細菌重要的毒力基因,可增強布魯氏菌胞內(nèi)存活能力。同時已有文獻報道virB和vjbR關(guān)系密切,相互影響。那么vjbR是否與細菌毒力表型相關(guān)?是否可以調(diào)控virB從而影響布魯氏菌胞內(nèi)生存?vjbR的調(diào)控機制是怎樣的呢?這些問題的回答,有利于解釋和深入探討布魯氏菌胞內(nèi)生存機制和致病機制。 為研究布魯氏菌QS系統(tǒng)調(diào)控蛋白VjbR與布魯氏菌毒力和胞內(nèi)生存的關(guān)系,闡明其調(diào)控機制,本研究首先構(gòu)建vjbR基因缺失突變株和互補株。通過對突變株、互補株和野生株生長曲線的觀察,發(fā)現(xiàn)野生株的生長速度較突變株和互補株快,提示vjbR可能通過調(diào)控特定基因而正調(diào)控布魯氏菌的生長。胞內(nèi)存活及小鼠體內(nèi)生存等表型實驗顯示,vjbR突變株雖然可以入侵巨噬細胞并在小鼠的脾臟和肝臟細胞內(nèi)生存,但生存能力減弱,同時,我們在體外模擬了巨噬細胞內(nèi)的多種脅迫條件,如高鹽、高滲、酸、熱休克和氧壓力等,與野生株和互補株相比,突變株在這些刺激條件下的生存率都有不同程度的降低,表明vjbR對于布魯氏菌適應胞內(nèi)惡性環(huán)境、抵抗環(huán)境壓力、建立慢性感染是必需的。與此同時,我們又研制了布魯氏菌全基因組DNA芯片。利用全基因組芯片,比較分析了vjbR突變株與16M野生株轉(zhuǎn)錄譜差異,鑒定vjbR調(diào)控的靶基因,并對這些基因的功能進行詳細闡述,為QS調(diào)控網(wǎng)絡的研究提供靶標基因。本研究中,我們對布魯氏菌QS系統(tǒng)在毒力中發(fā)揮的作用及其調(diào)控機制進行了詳細的探討,為布魯氏菌治病機制的研究提供大量有價值的信息。
[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.
【學位授予單位】：吉林大學
【學位級別】：博士
【學位授予年份】：2009
【分類號】：R378.51
本文編號：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.
【學位授予單位】：吉林大學
【學位級別】：博士
【學位授予年份】：2009
【分類號】：R378.51
【引證文獻】
相關(guān)碩士學位論文 前1條
1 王同坤;布魯氏菌非編碼小RNA的預測、鑒定及BSR-16的功能分析[D];吉林大學;2011年
，本文編號：2421666
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