【作者】 张义全；

【导师】 宋亚军； 周冬生；

【作者基本信息】 中国人民解放军军事医学科学院 ， 军事预防医学， 2011， 硕士

【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Mg²⁺还是低Mg²⁺以及酸性环境下,RovA对psaA的转录都具有促进作用,但是在低Mg²⁺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Mg²⁺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Mg²⁺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。更多还原

【Abstract】 Yersinia pestis is the causative agent of plague, an acute zoonotic infection fatal to humans. During the transmission cycles and pathogenesis processes, Y. pestis will encounter multiple environmental adverse simulation, such as antibiotic peptides, temperature changing, pH and osmotic pressures. Y. pestis can sense and response to these diverse environmental signals, then initialize self-regulation mechanisms secrete virulence factors and archive successful survive. One of the most important self-regulation mechanisms is that regulators response the environmental signals and regulate the transcription of their target genes. In Y. pestis, both PhoP and RovA are the virulence regulators, and pH6 antigen is an important virulence related factor, but the relationship between them is still unknown.PhoP is the regulator protein of two-component system PhoP-PhoQ. The PhoP-PhoQ system can response to the signals of low-magnesium, mild acid pH and antibacterial peptides, and then activate PhoP protein, the activated-PhoP can activate or repress the target genes’transcription. Our previous results in Y. pestis revealed that, PhoP is a globe regulator. For bacteria growing in low-magnesium culture media, PhoP regulate at least 706 targets at transcriptional level, including psa locus, phoP-phoQ system and rovA.It has been established that RovA is a virulent regulator, as the virulence of Y. enterocolitica rovA null mutant strain to mice declined significantly compared to wild strain. RovA can regulate the expression of target genes through binding to their promoter regions in dimeride form. In Y. enterocolitica and Y. pseudotuberculosis, RovA not only stimulate the transcription of inv, but also other virulence related genes and itself as well. Our study based on microarray transcriptional analysis indicated that, dozens of genes(including the gene cluster of pH6 antigen, psa locus) were regulated by RovA at transcriptional level. Although RovA is proven a global regulator, there is no data about its regulate mechanism in Y. pestis by now.The pH6 antigen is an important virulence related factor in Y. pestis, and it is closely related to bubonic plauge. pH6 antigen in Y. pestis is encoded by psaEFABC operon. Cluster psaABC encode subunit protein PsaA, putative chaperone PsaB and membrane protein PsaC respectively. Meanwhile PsaEF may get involved in the transcriptional regulation of psaA. Expresson of pH6 antigen is affected by both temprature and pH: it won’t get expression at the temperature lower than 34℃and will have the highest expression at pH6.0.According to microarray and bioinformatics analysis, the transcription of psaA, psaE, phoPQ operon and rovA can be regulated by PhoP, meanwhile rovA and psaA, psaE may be regulated by RovA. The aim of this research is to clarify the regulation mechanism of PhoP and RovA to their possible target genes.We firstly obtained Y. pestis phoP and rovA null mutant, by using Red-based one-step gene inactivite system. Then we employed the E. coli BL21λDE3 to express recombinant PhoP and RovA proteins. Then we applied the classical melocular experiments, such as Electrophoretic mobility shift assays (EMSA), DNaseⅠfootprinting, lacZ fusion assay and primer extension, to decipher the regulate machanism of PhoP or RovA to it’s own targets.We found out that, the transcription of psaA, psaE and rovA were all inhibited by PhoP under low-magnesium and medial log-phage conditions; meanwhile it’s own operon (YPO1635 and phoP) were both activated. For pH5.8 and mid-log phase conditions, the transcription of psaA was repressed by PhoP, but other targets were no affected. For mid-log phase conditions, RovA activates the transcription of psaA, psaE and rovA. We also found out that the transcription of psaA was regulated by acid as the expression of pH6 antigen genes was higher in pH5.8 than in pH 7.2; while temperature had no influence on the expression of psaA.For rovA, we identified two transcription starts in it’s promoter region, which were named P1 and P2 respectively. P1 was much more closer to the translation start“ATG”than P2. P1 was PhoP-repressed and RovA-activited, while P2 was only RovA-dependent. DNaseⅠfootprinting indicated that there are two sites (named site1 and site2) for RovA in rovA promoter region, and RovA had a much higher affinity to site1 than site 2, while site1 located upstream close to P2. We postulated that rovA would be activated from P2 when RovA binding to site 1, but inhibited when RovA binding to site 2, as site 2 was downstream of P1. This hyphothsis proposed RovA had double effects on the transcription of rovA.For psaE, the binding sites of PhoP and RovA were identied, and three transcriptional starts were also found. PhoP binding site in psaE almost covered all the three transcriptional starts, while RovA binding site located upstream of the PhoP’s, farther to the transcription starts. According to in vivo experimentsresults, the transcription of psaE was inhibitated by PhoP, but activiated by RovA under special conditions.Binding sites of PhoP and RovA in the promoter region of psaA overlaps, and both sites were downstream of transcriptional start. Results from in vivo experiments showed that, in low-magnesium or mild acid pH culture media, RovA activiated psaA transcription, while PhoP inhibitated it, So we can induced that PhoP and RovA co-regulated the transcription of psaA through competing for the same binding site of psaA promoter.Under low-magnesium condition, the activiation of the expression of YPO1635 by PhoP has been demonstarted in other papers.In this study, we confirmed this phenomenon. We also found out that PhoP activated self-transcription. For the two transcriptional starts identified in phoP promoter, namely P1 and P2, but only P2 was PhoP-dependent in mid-log phase.In shortly, this paper applied molecular and biochemical test methods, firstly discovered the transcriptional regulation cycle of PhoP-RovA-psa, that RovA activated the transcription of psa locus and rovA, and under low-magnesium condition, PhoP inhibited the transcription of psa locus and rovA, but activated its’own operon, PhoP can sense and response to acid signal to inhibited the transcription of psaA; The transcription of psaA was also regulated by acid , one may reap more as the expression of pH6 antigen genes were higher in pH5.8 than in pH 7.2, however, the temperature had no influence on the expression of psaA.更多还原