QS系统VjbR在布鲁氏菌胞内生存中作用的研究
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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。
Brucellosis, which is caused by Brucella, is one of the most important bacterial zoonoses endemic in the world, especially in developing countries. These pathogens can affect a broad range of mammals and cause serious economic losses. The unique pathogenicity is the research focus on this pathogen. Brucella is an intracellular bacterium, and the virulence depends upon its ability to survive and replicate within host cells. Quorum sensing is a cell-density dependent global regulation system, which is used for cell to cell communication mediated by the signal molecule, N-acyl-homoserine lactone (AHL). However, the molecular mechanisms underlying regulation remain unclear.
In the present study, homologous recombination was used to construct the vjbR deletion mutant, and then, the survival in macrophages and in mice of wild-type and mutant were compared to define related phenotypes affected by vjbR. The transcriptome of the mutant was compared to the wild-type strain to define the differentially transcribed genes, which may be regulated by vjbR. By combining the function information of the differentially transcribed genes and the alterations of the phenotypes in the mutant, the regulation mechanism was putatively explained.
To define the vjbR related phenotypes and the target genes regulated by vjbR, the vjbR mutant and its complementary strain were to be constructed. Firstly, the kanamycin gene of pBBR1MCS-2 was amplified by PCR method and cloned at the multicloning site of pUC19 to generate a new suicide plasmid pUC19K, which was then used to construct the deletion mutant strain of 16M to generate16MΔvjbR. And then, the ORF of vjbR gene was cloned into pMD18-T vector to construct the complementary plasmid, which was then transformed into 16MΔvjbR to generate the complementary strain 16MΔvjbR-C. Semi-quantitative reverse transcription PCR results showed that transcription of the vjbR genes was detected in the wild-type and complementary strain, but not in the vjbR mutant, indicating that vjbR was successfully inactivated in the mutant and restored in the complementary strain.
Then, the related intracellular survival phenotypes of wild-type, mutant and complementary strain were compared. Growth curve assays in vitro showed that the wild-type virulent strain 16M displayed higher growth rate than the vjbR mutant and the complementary strain at logarithmic phase. This implied that QS positively regulate growth of Brucella by regulating other related genes. Survival in macrophages and in mice showed that the mutant could invade the macrophages but the disruption of vjbR led to a decreased survival in macrophages and a drastic reduction in spleen or liver colonization in mice, implying that vjbR is essential for intracellular survival and chronic infection of Brucella. Survival under stress conditions showed that the vjbR mutant was sensitive to the stress conditions which simulated intracellular environments, including high salt, high osmosis, low pH, heat shock and oxidative stress. The vjbR mutant was more sensitive to high salt, osmotic stress, polymyxin B and sodium deoxycholate, indicating that vjbR is important for membrane integrity, and resistance to hostile environments. The vjbR mutant was also sensitive to oxidative and acidic stress, which simulated intracellular environment, indicating its role in adaptation to these harsh environments.
After the elucidation of the intracellular survival phenotypes, a comparative transcriptome analysis was used to define the genes regulated by vjbR, with the aims to explain the possible regulation mechanisms of vjbR at transcription level. As a tool to perform the comparative transcriptome analysis, the non-redundant whole-genome DNA microarrays were developed based on the genomic sequences of B. melitensis strain 16M. A total number of 3968 genes was amplified by PCR and printed in duplicate onto glass slides. Fluorescently labeled probes were prepared by priming of genomic DNAs or cDNAs with random hexamers and extension with Klenow. Labelled DNAs or cDNAs were hybridized to the microarrays by methods of two-fluorescence comparative hybridization. The results were validated with semi-quantitative reverse transcription PCR.
With the DNA microarray, we compared the transcription profiles of the vjbR mutant to that of 16M. Firstly, quantitative RT-PCR was used to determine the stress condition under which the transcription of vjbR was greatly activated. The results showed that the vjbR was greatly induced under acidified GEM pH4.0 for 2h. Then, comparative transcription analysis was performed under this condition. By a 2 fold change criteria, sixty three genes, including those encoding the type IV secetion system, energey/lipid/amino acid/carbohydrate/ion metabolism, cell envelop and hepothetical protein, were differentially transcribed in vjbR mutant.
The results above indicated that the vjbR mutant showed several phenotype changes, reduced survival in the macrophage and mice, and further comfirmed its role in intracellular survival by affecting related phenotypes. Results from comparative transcription profiles showed that the vjbR may contribute the adaptation of Brucella to hostile environments and survival in the host cell by regulating the expression of related genes. These results expand our knowledge about roles of vjbR and molecular mechanism of Brucella intracellular survival.
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。
Brucellosis, which is caused by Brucella, is one of the most important bacterial zoonoses endemic in the world, especially in developing countries. These pathogens can affect a broad range of mammals and cause serious economic losses. The unique pathogenicity is the research focus on this pathogen. Brucella is an intracellular bacterium, and the virulence depends upon its ability to survive and replicate within host cells. Quorum sensing is a cell-density dependent global regulation system, which is used for cell to cell communication mediated by the signal molecule, N-acyl-homoserine lactone (AHL). However, the molecular mechanisms underlying regulation remain unclear.
In the present study, homologous recombination was used to construct the vjbR deletion mutant, and then, the survival in macrophages and in mice of wild-type and mutant were compared to define related phenotypes affected by vjbR. The transcriptome of the mutant was compared to the wild-type strain to define the differentially transcribed genes, which may be regulated by vjbR. By combining the function information of the differentially transcribed genes and the alterations of the phenotypes in the mutant, the regulation mechanism was putatively explained.
To define the vjbR related phenotypes and the target genes regulated by vjbR, the vjbR mutant and its complementary strain were to be constructed. Firstly, the kanamycin gene of pBBR1MCS-2 was amplified by PCR method and cloned at the multicloning site of pUC19 to generate a new suicide plasmid pUC19K, which was then used to construct the deletion mutant strain of 16M to generate16MΔvjbR. And then, the ORF of vjbR gene was cloned into pMD18-T vector to construct the complementary plasmid, which was then transformed into 16MΔvjbR to generate the complementary strain 16MΔvjbR-C. Semi-quantitative reverse transcription PCR results showed that transcription of the vjbR genes was detected in the wild-type and complementary strain, but not in the vjbR mutant, indicating that vjbR was successfully inactivated in the mutant and restored in the complementary strain.
Then, the related intracellular survival phenotypes of wild-type, mutant and complementary strain were compared. Growth curve assays in vitro showed that the wild-type virulent strain 16M displayed higher growth rate than the vjbR mutant and the complementary strain at logarithmic phase. This implied that QS positively regulate growth of Brucella by regulating other related genes. Survival in macrophages and in mice showed that the mutant could invade the macrophages but the disruption of vjbR led to a decreased survival in macrophages and a drastic reduction in spleen or liver colonization in mice, implying that vjbR is essential for intracellular survival and chronic infection of Brucella. Survival under stress conditions showed that the vjbR mutant was sensitive to the stress conditions which simulated intracellular environments, including high salt, high osmosis, low pH, heat shock and oxidative stress. The vjbR mutant was more sensitive to high salt, osmotic stress, polymyxin B and sodium deoxycholate, indicating that vjbR is important for membrane integrity, and resistance to hostile environments. The vjbR mutant was also sensitive to oxidative and acidic stress, which simulated intracellular environment, indicating its role in adaptation to these harsh environments.
After the elucidation of the intracellular survival phenotypes, a comparative transcriptome analysis was used to define the genes regulated by vjbR, with the aims to explain the possible regulation mechanisms of vjbR at transcription level. As a tool to perform the comparative transcriptome analysis, the non-redundant whole-genome DNA microarrays were developed based on the genomic sequences of B. melitensis strain 16M. A total number of 3968 genes was amplified by PCR and printed in duplicate onto glass slides. Fluorescently labeled probes were prepared by priming of genomic DNAs or cDNAs with random hexamers and extension with Klenow. Labelled DNAs or cDNAs were hybridized to the microarrays by methods of two-fluorescence comparative hybridization. The results were validated with semi-quantitative reverse transcription PCR.
With the DNA microarray, we compared the transcription profiles of the vjbR mutant to that of 16M. Firstly, quantitative RT-PCR was used to determine the stress condition under which the transcription of vjbR was greatly activated. The results showed that the vjbR was greatly induced under acidified GEM pH4.0 for 2h. Then, comparative transcription analysis was performed under this condition. By a 2 fold change criteria, sixty three genes, including those encoding the type IV secetion system, energey/lipid/amino acid/carbohydrate/ion metabolism, cell envelop and hepothetical protein, were differentially transcribed in vjbR mutant.
The results above indicated that the vjbR mutant showed several phenotype changes, reduced survival in the macrophage and mice, and further comfirmed its role in intracellular survival by affecting related phenotypes. Results from comparative transcription profiles showed that the vjbR may contribute the adaptation of Brucella to hostile environments and survival in the host cell by regulating the expression of related genes. These results expand our knowledge about roles of vjbR and molecular mechanism of Brucella intracellular survival.
引文
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