整体调控子PhoP直接调控鼠疫耶尔森氏菌胞内生存能力的研究

发布时间：2018-01-09 07:12

本文关键词：整体调控子PhoP直接调控鼠疫耶尔森氏菌胞内生存能力的研究　出处：《中国人民解放军军事医学科学院》2006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，是由鼠疫耶尔森氏菌(以下简称鼠疫菌)引起的。机体感染早期，鼠疫菌能在巨噬细胞内存活并繁殖，是鼠疫菌致病的关键阶段。PhoP-PhoQ是一种二元调控系统(two-component system)，它可以调控细菌的毒力，参与细菌对Mg~(2+)限制性生长环境的适应，而且还可以调节几种革兰氏阴性细菌的许多细胞活性。研究发现△phoP突变株对小鼠J774巨噬细胞感染力下降，对巨噬细胞杀(抑)菌机制(酸胁迫、过氧化氢刺激、高渗胁迫、抗菌肽作用)敏感性大大增强。虽然毒力和生化表型实验证明了PhoP-PhoQ控制着鼠疫菌胞内寄生能力，但是PhoP-PhoQ如何控制这个过程的机制至今尚未阐明。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phoP基因，进而利用鼠疫菌全基因组DNA芯片进行基因转录调控研究。通过比较△phoP突变株和野生株在低镁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PhoP调控元，这一分析可从全基因组水平筛选出所有受PhoP-PhoQ直接或间接调控的靶基因。然后，利用经典分子生化实验和生物信息学分析，深入探究二元调控系统PhoP-PhoQ转录调控的分子机制。用凝胶迁移实验明确46个操纵子／基因直接受PhoP的转录调控。这些基因有的受PhoP的上调，有的受其下调，包括PhoP调控的各方面功能的基因。PhoP正调控mgtCB，后者可以调节Mg~(2+)的转运；通过调节氧化应激基因、分子伴侣和ATP-依赖性蛋白酶基因以及其他应激基因来参与菌体的应激防御机制；通过正调控glgPACXB操纵子来参与恢复高渗条件下细胞浆的体积、正调控pmrHFIJKLM和ugd基因来实现脂质A的氨基阿拉伯糖修饰、调控代谢相关基因来参与鼠疫菌能量代谢、大分子和小分子的降解以及氨基酸、嘌呤、嘧啶、核苷酸、辅基和脂肪酸生物合成等。值得注意的是，鼠疫菌的PhoP能对包括其自身在内的八个调节基因，phoP、crp、lacI、phoB、cyaA、ntrB、slyA和glnK，进行直接调控从而发挥更大的级联作用。因此，二元调控系统PhoP-PhoQ应当是鼠疫菌的一个整体调控子。为了明确PhoP的精细调控机制，本研究对26个凝胶迁移实验阳性的基因的启动子进行了足迹实验，得到了25个PhoP结合保护区域，在这个区域内存在着PhoP box，PhoP蛋白调控靶基因主要是通过结合各靶基因启动子内的PhoP box，调控各基因的转录。用motif sampler软件对这25个结合保护区域进行同源比对，并从统计学角度分析了18个位置上的碱基分布情况，最终将鼠疫菌的PhoP box归纳为(T／G)(A／G)TTTA(A／T)七核苷酸同向重复序列。但PhoP box存在
[Abstract]:Yersinia pestis is an ancient natural disease caused by Yersinia pestis. In the early stage of infection, Yersinia pestis can survive and reproduce in macrophages. PhoP-PhoQ is a two-component system, which can control the virulence of bacteria. To participate in the adaptation of bacteria to Mg~(2) restrictive growth environment. And it can also regulate the cell activity of several Gram-negative bacteria. It was found that the phoP mutant decreased the infectivity of J774 macrophages and killed (inhibited) the macrophages (acid stress). The sensitivity of hydrogen peroxide stimulation, hyperosmotic stress and antimicrobial peptide was greatly enhanced, although virulence and biochemical phenotype experiments proved that PhoP-PhoQ controlled the parasitic ability of Yersinia pestis. But the mechanism by which PhoP-PhoQ controls this process has not yet been clarified. In this study, the phoP gene of Yersinia pestis was replaced by one step mutation technique based on Red system. Then the gene transcriptional regulation of Yersinia pestis was studied by using the whole genome DNA chip. By comparing the transcription level of phoP mutant and wild strain under low magnesium condition, the up-regulated and down-regulated genes were defined. These genes form the PhoP regulator, an analysis that selects all target genes directly or indirectly regulated by PhoP-PhoQ at the genome level. By means of classical molecular biochemistry experiment and bioinformatics analysis, the molecular mechanism of PhoP-PhoQ transcriptional regulation was deeply explored. Gel migration assay showed that 46 operons / genes were directly regulated by the transcription of PhoP. Some of these genes were up-regulated by PhoP and others were down-regulated. The gene. PhoP, which includes all aspects of PhoP regulation, regulates the transport of Mg~(2. By regulating oxidative stress genes, molecular chaperones, ATP-dependent protease genes and other stress genes, they participate in the stress defense mechanism of bacteria. Through the positive regulation of glgPACXB operon to participate in the restoration of the cytoplasm volume under hypertonic condition, and the regulation of pmrHFIJKLM and ugd genes to achieve the amino arabinose modification of lipid A. Regulation of metabolism-related genes to participate in Yersinia pestis energy metabolism, degradation of macromolecules and small molecules, amino acid, purine, pyrimidine, nucleotide, cogroups and fatty acid biosynthesis. The PhoP of Yersinia pestis can affect the eight regulatory genes, including its own, Phosphorus cryptifolia and glnK. Therefore, the dual control system PhoP-PhoQ should be an integral regulator of Yersinia pestis. In order to elucidate the fine regulation mechanism of PhoP, the promoter of 26 positive genes for gel migration test was studied by footprint test, and 25 PhoP binding protection regions were obtained. In this region, the target gene of PhoP boxphop protein is regulated mainly by PhoP box in the promoter of each target gene. The gene transcription was regulated by motif sampler software. The 25 protected regions were compared with each other, and the distribution of the bases in 18 sites was analyzed from the point of view of statistics. Finally, the PhoP box of Yersinia pestis was summed up as the homozygote sequence of T / G box. But PhoP box existed.
【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】：2006
【分类号】：R378

【引证文献】