鼠疫菌毒力调节因子Hfq及CRP作用机制的研究

发布时间：2019-05-19 06:17

【摘要】：背景：鼠疫是由鼠疫耶尔森氏菌引起的自然疫源性疾病，在人类历史上曾经多次暴发流行。在感染宿主的过程中，鼠疫菌必须及时调整以适应外部环境压力同时维持其毒力和致病性，这些调整过程伴随着鼠疫菌基因调控的变化。毒力调节因子通过毒力调节网络对各个靶基因的表达进行上调或下调，其中调节因子Hfq和CRP的调节过程在毒力调节过程中起着特别重要作用。Hfq是sRNA分子伴侣，介导sRNA与靶mRNA之间的相互作用，基因hfq的缺失会导致鼠疫菌毒力的下降。环化腺苷单磷酸受体蛋白（CRP）在碳源代谢产物抑制过程中起重要作用，是鼠疫菌在宿主体内生存繁殖过程中根据外部碳源调整自身代谢途径以及能量代谢过程的核心因子；Crp基因的缺失会导致鼠疫菌毒力的减弱。本实验希望对鼠疫菌毒力调节的两个因子Hfq和CRP的作用机制进行研究。方法：采用基于-Red重组系统的基因突变方法，分别突变鼠疫菌基因hfq和crp，构建鼠疫菌hfq突变株（hfq）和crp突变株（crp）。对Hfq研究方面，，通过表型实验验证鼠疫菌hfq基因的缺失对生物膜形成的总体影响。通过-半乳糖苷酶活性实验反映Hfq对各靶基因转录启动水平的调节情况。通过引物延伸实验反映各靶基因转录至mRNA水平受Hfq影响。在比较hms各个基因翻译至蛋白水平的变化方面，先将hms各个基因重组表达并免疫兔子得到多抗，再进行western blot实验。使用液相色谱-质谱联用技术分析了鼠疫菌野生株、 hfq株和C-hfq株胞内c-di-GMP的水平。对CRP研究方面，通过生长曲线和不同的培养基比较了鼠疫菌野生株和crp生长情况的差异。通过引物延伸实验和-半乳糖苷酶活性实验，验证了CRP对其自身基因和编码腺苷酸环化酶的基因（cyaA）转录调节作用。使用电泳迁移实验（EMSA）和DNA酶Ⅰ足迹实验得出了CRP对其自身基因和编码腺苷酸环化酶基因的结合位点。比较了鼠疫菌野生株和crp株胞内cAMP的浓度。结果：对于Hfq因子，在质粒pCD1存在的情况下，Hfq对生物膜形成过程起正调节作用；而在缺失质粒pCD1的情况下，Hfq对生物膜形成过程起负调节作用。-半乳糖苷酶活性实验表明，Hfq对hmsP基因的转录启动为负调节作用，对hms其他基因在转录启动没有作用。引物延伸实验表明，在转录后的mRNA水平Hfq对hmsHFRS，hmsT基因是正调节，对hmsP基因是负调节，对hmsD基因无调节。Western blot实验表明，在转录后蛋白水平Hfq对hms各基因调节结果同其在mRNA水平的调节结果。对于CRP因子，基因crp的缺失导致鼠疫菌生长受到影响， crp株生长速率以及平台期菌浓度均低于野生株。-半乳糖苷酶活性实验表明，CRP对自身基因的转录启动无调节，而对基因cyaA负调节。引物延伸实验显示，CRP对基因crp的mRNA无调节，而对基因cyaA负调节。基因crp的转录起始位点为A（-285），基因cyaA的转录起始位点为G（-133）。电泳迁移阻滞实验和DNA酶I足迹实验证明，CRP对基因cyaA有直接结合作用。胞内环化腺苷单磷酸（cAMP）浓度的测定进一步证实CRP对基因cyaA的负调节作用。结论：本研究对鼠疫菌中两个毒力调节因子Hfq和CRP在各自的毒力调节途径——生物膜的形成和腺苷酸环化酶调节，分别进行了研究。结果表明：质粒pCD1的存在对Hfq调节生物膜的过程有较大影响，在质粒存在的情况下Hfq对生物膜是正调节，在质粒缺失的情况下Hfq对生物膜是负调节。Hfq通过转录后方式正调节hmsHFRS，hmsT，通过转录和转录后方式负调节hmsP，共同调节生物膜形成，hmsD在本实验条件下不参与Hfq调节生物膜的过程。CRP对其自身基因无调节，并且不作用于自身基因启动子区。CRP结合于编码腺苷酸环化酶的基因（cyaA）启动子区的-10序列附近，并且抑制其转录。CRP通过作用于腺苷酸环化酶基因来抑制cAMP的产生，这代表了鼠疫菌中CRP负反馈自调节的一种机制。
[Abstract]:BACKGROUND: The plague is a natural infectious disease caused by Yersinia pestis. In the process of infecting the host, the Yersinia pestis must be adjusted in time to adapt to the external environment pressure while maintaining its virulence and pathogenicity, which are accompanied by changes in the regulation of the Yersinia pestis genes. The regulation factors of the regulation factors, such as Hfq and CRP, play a particularly important role in the regulation of virulence. Hfq is a sRNA molecular chaperone, which mediates the interaction between sRNA and the target mRNA, and the deletion of the gene hfq results in a decrease in the virulence of the Yersinia pestis. The cyclic adeno-monophosphate receptor protein (CRP) plays an important role in the inhibition of carbon source metabolism products, and is a core factor for the regulation of the metabolic pathway and energy metabolism of the Yersinia pestis according to the external carbon source in the survival and reproduction process of the host. The deletion of the Crp gene can lead to a decrease in the virulence of the Yersinia pestis. The effects of two factors, such as Hfq and CRP, on the virulence regulation of Yersinia pestis were studied in this experiment. Methods: The hfq and crp mutants of Yersinia pestis (hfq) and crp mutants (crp) were constructed by mutation of hfq and crp of Yersinia pestis by mutation method based on-Red recombinant system. In the aspect of hfq, the total shadow of the formation of the biofilms by the deletion of the hfq gene of the yersinia pestis was verified by a phenotypic experiment. The response of Hfq to the transcriptional initiation level of each target gene was reflected by the activity of the-galactooligosaccharide. In which the transcription of each target gene to the mRNA level is reflected by the Hfq shadow by a primer extension experiment. In response to that change of the translation of the individual gene of hms to the level of the protein, the hms gene is recombinantly express and the rabbit is immunized to obtain multi-resistance, and western blot is carried out. The c-di-GMP water of the wild strains, hfq and C-hfq strains of Yersinia pestis were analyzed by liquid chromatography-mass spectrometry. In that study of CRP, the difference between the growth of the wild strain and the crp of Yersinia pestis was compared by the growth curve and the different culture medium. The gene (cyaA) transcriptional regulation of CRP on its own gene and the encoding of the adenoid acid cyclase was verified by the primer extension experiment and the activity of the-galactosyenase. Using an electrophoretic mobility experiment (EMSA) and a DNA enzyme I footstep experiment, the binding site of CRP to its own gene and the gene of the encoded adenoid acid cyclase was obtained. Points. The concentration of cAMP in the wild and crp strains of Yersinia pestis was compared. Results: In the presence of pCD1 of plasmid pCD1, Hfq plays a positive role in the process of biofilm formation. In the absence of plasmid pCD1, Hfq has a negative effect on the process of biofilm formation. The results showed that the transcription of hmsP gene was negatively regulated by Hfq, and the other genes of hms were not activated by transcription. The primer extension experiment shows that the hmsHFRS and hmsT gene are positive and the hmsP gene is negative regulated, and the hmsD gene is expressed by the primer extension experiment. The results of Western blot show that the regulation of hms of hms in the post-transcriptional protein level (hfq) is related to the level of mrna. The results showed that, for CRP, the deletion of gene crp results in the growth of Yersinia pestis, the growth rate of crp and the concentration of bacteria in plateau. Wild plant.-Galactomase activity experiment shows that CRP has no regulation on the transcription initiation of its own gene, and the gene cyaA Negative regulation. The primer extension experiment shows that the CRP has no regulation of the mRNA of the gene crp, and the gene cyaA The transcription initiation site of the gene crp is A (-285), and the transcription initiation site of the gene cyaA is G (-1). 33). The experiment of electrophoretic mobility block and the experimental results of DNA enzyme I have shown that CRP has a direct binding on the gene cyaA. The determination of the concentration of intracellular cyclized adenosine phosphate (cAMP) further confirmed the negative regulation of the CRP to the gene cyaA. The results showed that the two virulence regulation factors, Hfq and CRP in Yersinia pestis, were regulated by the respective virulence regulation pathway _ biofilm formation and the regulation of the adenoid acid cyclase, respectively. The results showed that the existence of plasmid pCD1 has a great effect on the process of the regulation of the biological membrane of Hfq. In the presence of the plasmid, the Hfq is under the regulation of the biological membrane, and the Hfq is positive in the case of the deletion of the plasmid. The membrane is negative regulation. hmshfrs and hmsT are regulated by the post-transcriptional mode, hmsP is regulated by transcription and post-transcriptional regulation, and the formation of biofilms is co-regulated. hmsD is not involved in the regulation of hfq under the experimental conditions. The CRP has no regulation on its own gene and does not act on its base. As a result of the promoter region. Inhibition of its transcription. CRP inhibits the production of cAMP by acting on the adeno-acid cyclase gene, which represents the negative feedback self-regulation of CRP in the Yersinia pestis.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R516.8

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