鼠疫耶尔森氏菌PhoP-RovA-psa转录调控环路研究
发布时间：2018-06-03 07:33
本文选题：鼠疫耶尔森氏菌 + 转录调控 ； 参考：《中国人民解放军军事医学科学院》2011年硕士论文
【摘要】：鼠疫是由鼠疫耶尔森氏菌(以下简称鼠疫菌)引起的一种自然疫源性疾病。鼠疫菌在传播环节中宿主动物的改变以及致宿主病变的过程中,受到诸多环境信号,比如抗菌肽、温度、pH、渗透压等的刺激,鼠疫菌能感应这种复杂的信号刺激并自我调节,产生毒力因子,最终使鼠疫菌得以存活。鼠疫菌自我调节机制中,转录调控子对靶基因在转录水平上的调节至关重要。PhoP和RovA是鼠疫菌重要的毒力调控因子,pH6抗原是鼠疫菌重要的毒力相关因子,它们之间联系至今并未被阐明。 PhoP是二元调节系统PhoP-PhoQ的调节蛋白,PhoP-PhoQ系统能够感应低Mg2+、微酸性环境以及抗菌肽的刺激信号,从而激活PhoP蛋白的转录调节活性,上调或下调靶基因的转录。本实验室前期研究发现,鼠疫菌PhoP蛋白是整体调控子,表达谱结果表明,在低Mg2+条件下,有706个基因的转录受PhoP的影响,包括pH6抗原基因、PhoP-PhoQ系统及rovA基因。 小肠结肠炎耶尔森氏菌rovA缺失株对小鼠毒力下降,表明RovA是其重要的毒力调控因子。RovA能以二聚化的形式结合到靶基因启动子区调控基因转录,在小肠结肠炎耶尔森氏菌和假结核耶尔森氏菌中,它不仅能激活侵袭因子基因inv的表达,还能激活自身的表达以及其他毒力相关基因的表达。在鼠疫菌中,也有学者进行了RovA的芯片表达谱分析,发现RovA是一个全局性毒力调控因子,能调控数十个基因的表达,包括pH6抗原基因,但是详细调控机制还没见报道。 鼠疫菌pH6抗原由基因簇psaEFABC编码,其中psaABC编码结构亚单位(A)及其伴侣蛋白(B)和膜引领蛋白(C);而psaEF表达产物可能对psaA的转录调控有关。pH6抗原最高表达是在环境pH=6、温度在34℃或更高条件下实现,且只有在34℃以上才能发挥毒力效应。pH6抗原是鼠疫菌的毒力相关因子,与腺鼠疫的发生密切相关。 根据表达谱和生物信息学预测,在转录水平上,PhoP对psaA、psaE、自身操纵子基因以及rovA可能具有调控作用,同时RovA对自身基因及psaA、psaE的转录也可能具有调节作用。本研究的目的在于阐明上述调控子对可能的靶基因的调控机制。 我们基于Red系统分别构建了鼠疫菌phoP和rovA基因的突变株,利用大肠杆菌BL21-DE3的蛋白表达系统分别获得His-PhoP和His-RovA重组蛋白,再利用凝胶阻滞实验(EMSA)、DNaseⅠ足迹实验、β-半乳糖苷酶报告基因融合实验(LacZ实验)以及引物延伸实验等来详细研究PhoP和RovA对各自靶基因的转录调控机制。 实验结果表明:在低Mg2+、对数生长中期条件下,在转录水平上PhoP抑制psaA、psaE、rovA的表达,而激活自身操纵子基因YPO1635、phoP的转录;pH5.8、对数生长中期条件下,在转录水平上PhoP抑制psaA的表达,而对其它所研究基因无影响;在对数生长中期,RovA能激活psaA、psaE以及rovA的转录;在对数生长中期,psaA的转录受酸的调节,pH5.8较pH7.2条件下高表达,但是温度对其无影响。 在rovA基因启动子区发现了两个转录起始位点,分别命名为P1和P2,其中P1靠近翻译起始位点,它的转录启动受PhoP的抑制而受RovA的激活,P2只受RovA的激活而不受PhoP的影响;DNaseⅠ足迹实验表明RovA对自身启动子区有两个亲和力不等的结合位点site1和site2,前者亲和力高于后者,site1位于P2之前,因此我们认为site1控制着P2的转录,site2位于P1之后,推测RovA与site2的结合对rovA的转录具有负反馈调节作用,即RovA的自调控是双重性质的。 在psaE启动子区分别得到了PhoP和RovA的结合位点,并发现了三个距离很近的转录起始位点,PhoP的结合位点几乎覆盖了这三个位点,RovA的结合位点紧挨着PhoP结合位点位于其上游而远离转录起始位点,体内实验也证明,在特定条件下PhoP对psaE转录具有抑制作用,而对rovA具有激活作用。 PhoP和RovA对psaA启动子的结合位点互相重叠,都位于转录起始位点之后,体内实验表明,无论是高Mg~(2+)还是低Mg~(2+)以及酸性环境下,RovA对psaA的转录都具有促进作用,但是在低Mg~(2+)和酸性条件下,PhoP对psaA的调控具有抑制作用,由此可见,PhoP和RovA通过竞争psaA启动子区相同的结合位点,共同调节psaA的转录。 低Mg~(2+)条件下,YPO1635受PhoP的正调控在其它文献资料中已经得到验证,本文也证明了这种调控关系,同时也证明了PhoP确实正调控自身的转录,但是所发现的两个转录起始位点P1和P2,在细菌生长对数中期时只有P2是PhoP依赖性的。 综上,本研究利用分子生化试验手段,首次揭示了鼠疫菌中的PhoP-RovA-psa转录调控环路:RovA正调控psa位点和自身的转录;低Mg~(2+)环境下,PhoP抑制psa位点和rovA的转录,而激活自身操纵子的转录,PhoP也能感应酸性信号,抑制psaA的转录;psaA的转录调节还受酸的调节,pH5.8较pH7.2条件下高表达,但是温度对其无影响。
[Abstract]:Yersinia pestis is a natural epidemic disease caused by the Yersinia pestis (hereinafter referred to as Yersinia pestis). In the process of the change of the host animal and the host disease, the Yersinia pestis is stimulated by a number of environmental signals, such as antimicrobial peptides, temperature, pH, osmotic pressure, and so on. Yersinia pestis can induce such complex signal stimulation and self - stimulation. I regulate, produce the virulence factor and eventually make the Yersinia pestis survive. In the self-regulation mechanism of Yersinia pestis, the regulation of the transcriptional regulator on the target gene at the transcriptional level is essential.PhoP and RovA are important virulence factors of Yersinia pestis, and the pH6 antigen is an important virulence related factor of Yersinia pestis, and the connection between them has not been elucidated.
PhoP is the regulation protein of the two element regulation system PhoP-PhoQ. The PhoP-PhoQ system can induce the low Mg2+, the micro acid environment and the stimulation signal of the antibacterial peptide, thus activating the transcriptional regulation activity of the PhoP protein and up regulating or down regulating the transcription of the target gene. Under low Mg2+ conditions, transcription of 706 genes is affected by PhoP, including pH6 antigen gene, PhoP-PhoQ system and rovA gene.
The virulence of the rovA strain rovA strain of enterocolitis shows that RovA is an important virulence regulator,.RovA, which can be combined in the form of dimerization to regulate gene transcription in the target gene promoter region. It not only activates the expression of the invasion factor gene inv in the enterocolitis of the enterocolitis, and in the Mycobacterium tuberculosis, Jerson S bacteria. It also activates its own expression and the expression of other virulence related genes. In Yersinia pestis, some scholars have carried out RovA chip expression analysis, and found that RovA is a global virulence regulator, which can regulate the expression of dozens of genes, including the pH6 antigen gene, but the detailed regulation mechanism has not yet been reported.
The pH6 antigen of Yersinia pestis is encoded by the gene cluster psaEFABC, in which psaABC encodes the structural subunit (A) and its chaperone protein (B) and membrane leading protein (C); while the psaEF expression product may express the highest expression of the.PH6 antigen on psaA's transcriptional regulation in environmental pH=6, the temperature is at 34 or more, and the virulence can be exerting only at 34 degrees centigrade. The effect.PH6 antigen is a virulence related factor of Yersinia pestis, and is closely related to the occurrence of bubonic plague.
According to the expression spectrum and bioinformatics, PhoP may play a regulatory role on psaA, psaE, the operon gene and rovA at the transcriptional level, and RovA may also regulate the transcription of its own genes and psaA and psaE. The purpose of this study is to elucidate the regulatory mechanism of the above regulator on the possible target genes.
The mutant strains of Yersinia pestis phoP and rovA gene were constructed based on the Red system. The recombinant protein of His-PhoP and His-RovA was obtained by the protein expression system of Escherichia coli BL21-DE3, and then the gel block experiment (EMSA), DNase I footprint experiment, beta galactosidase report gene fusion experiment (LacZ experiment) and primer extension experiment were used. To study the transcriptional regulation mechanism of PhoP and RovA on their respective target genes.
The experimental results showed that PhoP inhibited psaA, psaE, rovA expression at the transcriptional level at low Mg2+, at the transcriptional level, and activated the autoperon gene YPO1635, phoP transcription; pH5.8, at the medium-term condition of logarithmic growth, the PhoP inhibited psaA, but had no influence on other studied genes; in the middle period of logarithmic growth, Ro VA activates the transcription of psaA, psaE and rovA; in the middle of logarithmic growth, the transcription of psaA is regulated by acid, and pH5.8 is highly expressed in the pH7.2 condition, but the temperature has no effect on it.
Two transcriptional starting sites were found in the promoter region of the rovA gene, named P1 and P2, in which P1 was close to the translation initiation site, and its transcriptional initiation was activated by RovA, and P2 was only activated by RovA and was not affected by PhoP; DNase I footprint experiments showed that RovA had two unequal affinity to the self promoter region. Loci site1 and site2, the former is higher than the latter, and site1 is before P2. Therefore, we think site1 controls the transcription of P2. After site2 is located in P1, it is assumed that the combination of RovA and site2 has a negative feedback regulation on rovA transcription, that is, the self regulation of RovA is dual properties.
The binding sites of PhoP and RovA were obtained at the psaE promoter region, and three close transcriptional starting sites were found. The binding site of PhoP was almost covered by these three loci. The binding site of RovA was close to the PhoP binding site at its upstream and far away from the transcriptional starting site. In vivo experiments also proved that PhoP against PS under specific conditions. AE transcription has inhibitory effect on rovA.
The binding sites of PhoP and RovA overlapped with the psaA promoter, both at the starting site of the transcriptional starting point. In vivo experiments showed that RovA had a promoting effect on the transcription of psaA in both high Mg~ (2+) or low Mg~ (2+) and acidic environments, but under the low Mg~ (2+) and acid conditions, PhoP had an inhibitory effect on the regulation. OP and RovA co regulate psaA transcription by competing for the same binding sites in psaA promoter region.
Under the condition of low Mg~ (2+), the positive regulation of YPO1635 by PhoP has been verified in other literature. This paper also proves this regulation relationship, and also proves that PhoP does regulate its own transcription, but the two transcriptional starting sites, P1 and P2, are PhoP dependent in the mid-term logarithm of bacterial growth.
To sum up, this study revealed the PhoP-RovA-psa transcriptional regulation loop in Yersinia pestis for the first time: RovA is regulating the PSA locus and its own transcription. In low Mg~ (2+) environment, PhoP inhibits the transcription of PSA loci and rovA, and activates the transcription of the operon, PhoP can also induce acid signals, inhibit the transcription of psaA; psaA turn. The regulation of pH5.8 was also regulated by acid. The expression of pH5.8 was higher than that of pH7.2, but temperature had no effect on it.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R378
【共引文献】
相关期刊论文 前10条
1 宋干;;病毒性出血热新进展[J];传染病信息;2006年01期
2 庄汉澜;董梅;;炭疽免疫预防研究的现状及动向[J];传染病信息;2006年02期
3 张贵;张贵军;张芳;张雁冰;周方孝;Q，
本文编号：1971997
本文链接：https://www.wllwen.com/xiyixuelunwen/1971997.html
最近更新
教材专著
