鼠疫耶尔森氏菌毒力相关转录调控子Zur和PhoP的研究

发布时间：2018-01-04 08:41

本文关键词：鼠疫耶尔森氏菌毒力相关转录调控子Zur和PhoP的研究　出处：《中国人民解放军军事医学科学院》2008年博士论文　论文类型：学位论文

【摘要】： 鼠疫是一种古老的自然疫源性疾病,其病原菌鼠疫耶尔森氏菌(以下简称鼠疫菌)是多宿主寄生菌,体现在:鼠疫菌以蚤类为传播媒介寄居在特定的宿主(主要是啮齿动物),并在自然界宿主中造成鼠疫疫情的周期性爆发,该过程中偶然与患畜接触或被带菌跳蚤叮咬而感染到人。面对上述的复杂因素,鼠疫菌必须能够感应并快速适应每个环节的变化,才能在宿主体内存活并在自然界中传播最终致病,而每个适应性反应也必然伴随着鼠疫菌基因转录改变,这体现在:很多特定的调控子,分别感应特定的外界信号,介导特定基因(包括毒力基因)表达的上调和下调,最终组成复杂的毒力调控网络,控制鼠疫菌在媒介和贮存宿主中生存能力和致病力。病原菌从感应信号刺激到表达毒力因子的过程中,毒力调控子起着不可替代的作用。广谱或特异性的调控子,控制着毒力基因的表达;另外,若干个毒力调控基因的表达产物可组成一个复杂系统,执行毒力调控的功能,常称作毒力调控系统。一直作为研究热点的细菌毒力调控系统主要有QS系统、二元调控系统、Fur系统等等。这些系统又分成许多子调控系统,控制着不同类型毒力基因的表达,在细菌表现毒力的不同阶段起着调控作用。 Zur阻遏蛋白属于Fur家族,可以调控锌离子的转运,锌离子在所有生物细胞中是一种重要的蛋白质功能组分。PhoP-PhoQ是一种二元调控系统(two-component system),可以调控细菌的毒力,参与细菌对Mg2+限制性生长环境的适应,从而有利于鼠疫菌在巨噬细胞内的存活和繁殖。为了鉴定鼠疫菌毒力调控子Zur和PhoP的靶基因,尤其是直接调控的靶基因,并进一步研究Zur和PhoP对其下游直接靶基因的精细调控机制,本研究首先利用基于Red系统的一步法突变技术缺失替换鼠疫菌的zur和phoP基因,进而利用鼠疫菌全基因组DNA芯片进行基因转录谱分析。通过比较突变株和野生株在特定条件下的转录水平差异,界定表达上调和下调的基因,这些基因组成了Zur或PhoP调控元。这一分析可从全基因组水平筛选出所有受Zur或PhoP直接或间接调控的靶基因。转录谱的分析结果可以通过Real time RT-PCR进行验证。凝胶阻滞实验技术的应用,可以鉴定直接调控的靶基因,选择若干关键毒力相关基因,采用DNA酶I足迹实验技术、引物延伸实验技术,深入研究Zur和PhoP是如何直接调控这些基因的转录和表达,将毒力表型和分子实验数据结合起来,认识Zur和PhoP如何通过激活或抑制特定基因的表达,控制着鼠疫菌的宿主适应力和致病力。与野生株相比,敲除了zur基因的鼠疫菌在锌离子刺激下有154个基因发生了转录丰度改变,其中64个基因转录上调,90个基因转录下调。汇总文献预测的Zur结合位点,通过consensus-matrix和convert-matrix程序计算Zur结合序列每个位置四个碱基出现的权重,得到Zur结合序列的Matrix,进而用WebLogo软件展示序列logo,最终预测得到Zur结合box:GAAATGTTATAWTATAACATTTC。基于上述Zur基序分析和Zur调控子的转录谱分析,我们通过Matrix scan程序查找Zur保守基序,选取weight值较高的4个基因ykgM、znuC、znuA、astA进行进一步的分子生化实验。通过凝胶阻滞实验明确了ykgM、znuC、znuA 3个转录单元直接受Zur调控。为了明确Zur的精细调控机制,本研究对这三个转录单元的启动子进行了足迹实验,得到了精确的Zur结合保护区域,在这个区域内存在着预测的Zur box,Zur蛋白直接调控的这三个转录单元,其Zur box均覆盖-10序列,这样就阻碍了RNA聚合酶的结合,可能正因为如此,使Zur表现出对它们的抑制特性。对锌离子刺激下的野生株和?zur突变株同时进行引物延伸实验,不仅可以明确受Zur转录调控的鼠疫菌ykgM、znuC和znuA的转录起始位点,也可以确定Zur对其转录的影响。同时结合足迹实验得到的Zur结合位点,推断出每个启动子内的RNA聚合酶结合位点(-10序列)以及-35序列,丰富了Zur调控元启动子区的分子特性。前期对于?phoP突变株和野生株在低镁条件下的转录谱分析,已经初步界定了PhoP调控元,同样,我们用Real time RT-PCR对转录谱的分析结果进行验证。凝胶阻滞实验确定了30个转录单元直接受PhoP的转录调控,对其中17个基因的启动子区做了足迹实验,得到19个PhoP结合保护区域。通过生物信息学分析将鼠疫菌的PhoP box归纳为TGTTTAW七核苷酸同向重复序列,结合引物延伸实验得到的基因转录起始位点的位置信息,根据鼠疫菌的PhoP box在靶基因启动子内的位置以及是否含有-35序列,我们将PhoP启动子结构特征归纳为三类:PhoP结合位点在-35序列上游;PhoP结合位点覆盖-35序列;PhoP结合位点在-10序列的下游。本研究首次较全面地鉴定了鼠疫菌Zur和PhoP直接调控的调控元,同时从分子水平上明确了鼠疫菌Zur box和PhoP box的特性,探究了Zur和PhoP转录调控机制,为Zur和PhoP这类毒力调控子调控网络的构建,以及这些调控元功能的进一步明确提供了实验证据,这些机制的系统阐述将为鼠疫菌致病机制和疫苗的研究奠定重要基础。
[Abstract]:The plague is a kind of ancient natural foci of the disease, the pathogen of plague bacteria Jerson S (hereinafter referred to as Yersinia pestis) is multi host parasite, reflected in the plague fleas to the media resides in the specific host (mainly rodents), and in the natural host caused by periodic outbreaks of the plague. Accidental and patient contact or by infected flea bites to infect people in the process. In the face of the complex factors mentioned above, plague bacteria must be able to sense and adapt to the quick change of each link, in order to survive in the host body and the spread of final pathogenic in nature, and each adaptive response will inevitably accompanied by Yersinia pestis gene transcription changes. This is reflected in many specific promoter, respectively sensing specific external signals, mediated by specific genes (including virulence gene) expression up-regulated and down regulated, the final composition of virulence regulatory network complex To control the viability and pathogenicity of the Yersinia pestis in the medium and in the storage host.
The pathogenic bacteria from the induction signal process to stimulate the expression of virulence factors, virulence regulator plays an irreplaceable role. The global or specific regulators that control the expression of virulence genes; in addition, a number of virulence gene expression product can form a complex system, the implementation of the regulation of virulence function, often called the virulence regulation system. As the bacterial virulence regulation system of the main QS system, two element control system, Fur system and so on. These systems are divided into many sub control systems, control different types of virulence gene expression plays a role in different phase of bacterial virulence performance.
Zur repressor protein belongs to the Fur family, can regulate zinc ion transport, zinc ion is an important component of the.PhoP-PhoQ protein function is a two element control system in all living cells (two-component system), can control the virulence of bacteria, the bacteria involved in adaptation to Mg2+ restricted growth environment, and survival and breeding for Yersinia pestis in macrophages. In order to target gene identification of Yersinia pestis virulence regulators Zur and PhoP, especially the directly regulated target genes, and further study the fine regulation mechanism of Zur and PhoP on the downstream target gene, this study firstly uses the zur and phoP genes of one-step Red mutation deletion technology system based on the replacement of Yersinia pestis, Yersinia pestis and using whole genome DNA microarrays for gene transcription through spectrum analysis. The transcriptional difference comparison of mutant and wild-type strain under specific conditions, The definition of the expression of up-regulated and down regulated genes, these genes constitute a Zur or PhoP control element. This analysis can be screened out by all Zur or PhoP directly or indirectly regulate target genes from the whole genome level. The analysis results of transcriptional profiling can be verified by Real time RT-PCR. The application of experimental techniques of gel retardation, target gene identification of direct control, select some key virulence related genes, the DNA enzyme I footprinting experiment technology, experimental technique of primer extension, in-depth study of Zur and PhoP is how to directly regulate the transcription of these genes and expression of virulence phenotype and molecular experimental data combined with the knowledge of Zur and how PhoP expression through activation or inhibition of specific genes and control of plague host adaptation and virulence.
Compared with wild strains, strains with zur gene deletion of 154 genes transcript abundance changes under stimulation of zinc ions, including 64 up-regulated and 90 genes down regulated. Summary to predict Zur binding sites, through the consensus-matrix and convert-matrix program to calculate Zur weight with four nucleotide sequence for each position the Zur binding sequence of Matrix, and then use WebLogo software to show the sequence of logo, to predict the Zur combined with box:GAAATGTTATAWTATAACATTTC. spectrum analysis of the Zur transcription motif analysis and Zur regulator based on Matrix scan Zur by our program to find conserved motifs, we selected 4 genes ykgM, weight and higher znuC znuA. AstA molecular and biochemical experiments further. The gel retardation experiments confirmed that ykgM, znuC, znuA 3 transcription units directly regulated by Zur. In order to clear the fine tuning of Zur The control mechanism, the research on the promoter of the three transcription units of footprinting experiments, we obtain precise Zur combination of protected areas, in the area of memory in the prediction of Zur box, the three Zur protein directly regulate the transcription unit, the Zur box are covered with -10 sequence, thus impeding with RNA the polymerase, probably because of this, the Zur showed inhibitory properties of zinc ions on them. Under the stimulation of wild-type and mutant zur? And primer extension experiments can not only clear by Yersinia pestis ykgM Zur transcription, transcription initiation site of znuC and znuA, can also determine the effect of Zur on at the same time with the footprint of transcription. The experimentally obtained Zur binding sites inferred from each promoter within the RNA polymerase binding site (-10 sequence) and -35 sequence, enrich the molecular properties of Zur promoter regulatory elements.
For the pre? PhoP mutant and wild-type transcription in low magnesium under the condition of spectral analysis, has been defined in PhoP regulatory element, similarly, our results with Real time RT-PCR on the transcriptome verified. EMSA identified 30 transcription units by PhoP transcription regulation of the promoter. 17 genes do footprinting experiments, 19 PhoP with protection area. Through bioinformatics analysis of Yersinia pestis PhoP box will be summarized as TGTTTAW with seven nucleotide repeat sequences to the location information, gene transcription initiation site by primer extension obtained from the experiment, according to PhoP box of Yersinia pestis in target gene promoter in the position and the presence of -35 sequences, we will PhoP promoter structure is divided into three categories: PhoP binding sites in the upstream sequence of -35; PhoP binding sites covering -35 sequence; PhoP binding sites in -10 sequences Downstream.
This is the first study to comprehensively identify regulatory elements of Yersinia pestis Zur and PhoP directly regulated at the same time, from the molecular level to clear the characteristics of Yersinia pestis Zur box and PhoP box, Zur and PhoP on the mechanism of transcriptional regulation of Zur and PhoP, for the construction of this kind of virulence regulators, regulatory networks, and these regulatory elements to further clarify the function and provide experimental evidence, the system mechanism will lay an important foundation for the study of Yersinia pestis pathogenesis and vaccine.

【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R378

【共引文献】