结核分枝杆菌Wbl家族蛋白分析和WhiB3功能研究
发布时间：2018-09-10 12:18
【摘要】：结核分枝杆菌(Mycobacterium tuberculosis,Mtb)在感染过程中可以根据外界环境变化选择进行激活感染或潜伏感染。在调控感染的过程中,Mtb各个生理过程的调控是必需的,其中转录调控在生理过程调控中发挥不可或缺的作用,而转录调控蛋白通过不同调控机制参与到转录调控过程。为探讨Mtb转录调控蛋白的调控机制,本论文围绕Wbl家族蛋白,特别是Whi B3的功能与调控机制开展研究,主要包括以下5方面的内容:1.结核分枝杆菌调控蛋白的系统分析。基于单增李斯特菌(Listeria monocytogenes)抑制蛋白Mog R的抑制机理,构建了单体蛋白检测系统。Mog R蛋白包含N端的DNA结合结构域和C端的二聚化结构域。其中Mog R1-220能结合DNA,但不能抑制转录,基于此我们构建了将Mog R与调控蛋白融合的表达质粒。同时通过PCR将Mog R结合基序引入报告基因lac Z前的Ptac启动子中,得到报告质粒。系统构建后并对Mtb的调控蛋白进行了系统鉴定分析。154个调控蛋白中有109个可能是同源二聚体蛋白,32个可能为单体蛋白。在这些单体蛋白中,包括Wbl家族蛋白Whi B1-Whi B7。采用细菌双杂交系统检测Whi B1-Whi B7的自身相互作用,结果证明它们可能作为单体蛋白发挥调控功能。2.Wbl家族蛋白与σA相互作用的检测。通过蛋白共纯化、细菌双杂交和分枝杆菌蛋白互补等方法检测Wbl家族蛋白Whi B1-Whi B7与σA的相互作用,发现除Whi B5外都可以与σA相互结合。经过细菌双杂交系统和分枝杆菌蛋白互补系统检测,发现σA的R515位点影响与Whi B3、Whi B4、Whi B6和Whi B7的相互作用,而不能影响与Whi B1和Whi B2的相互作用。Whi B3在Mtb中发挥重要功能,以Whi B3为研究对象进行相互作用的进一步研究。采用细菌双杂交系统和蛋白共纯化分析,利用一系列突变体进行检测,发现σA的K508、P517和S518位点以及Whi B3的E81和E83会显著影响它们的相互作用。3.Mtb Whi B3的生理功能。在耻垢分枝杆菌(M.smegmatis,Ms)中过表达Mtb的Whi B3,不同生长期和缺氧条件取样,检测发现whi B3的转录在稳定期和缺氧时被诱导。在Ms野生型和Δwhi B3中诱导过表达Mtb的Whi B3后,表现为生长滞后、菌体加长和菌体沉降加快;而在σA的R515突变体菌株中,这些过表达效应消失。选取核糖体相关基因Msrps F和Msrps J为靶标进行研究,发现在稳定期添加诱导剂诱导Mtb的Whi B3表达没有影响这两个基因的转录,同时在MsΔwhi B3中检测不到明显的转录变化,表明不同时期过表达Mtb Whi B3产生不同的调控效应。4.Whi B3调控MMAR_3796-MMAR_3800操纵子的转录。在海分枝杆菌(M.marinum,Mm)突变whi B3后影响菌体色素的产生、对红细胞的裂解和溴化乙锭(EB)摄取,其中色素的产生与脂类合成相关。进一步在WT和Δwhi B3中检测Mm脂类合成相关基因的转录,发现MMAR_3796的转录受到明显调控。对整个操纵子基因的转录进行分析,发现该操纵子的5个基因均受到Whi B3的调控。5.酸性环境下whi B3转录激活调控机制。通过对whi B3的启动子(whi B3p)分析和体外EMSA实验,确定Pho P可以特异性结合whi B3p。回复Mm的pho P和whi B3双基因突变体,发现在酸性条件下Pho P被磷酸化后调控whi B3的转录。以耻垢分枝杆菌pho PR和whi B3双突变体为背景进行不同回复实验,发现酸性条件下whi B3的转录激活需要Pho R和Pho P的磷酸化过程。
[Abstract]:Mycobacterium tuberculosis (Mtb) can activate infection or latent infection according to the changes of external environment during infection. In the process of infection control, the regulation of various physiological processes of Mtb is necessary, in which transcriptional regulation plays an indispensable role in the regulation of physiological processes, and transcriptional regulation of eggs. White participates in the transcriptional regulation process through different regulatory mechanisms. To explore the regulatory mechanisms of Mtb transcriptional regulatory proteins, this paper focuses on the function and regulatory mechanisms of Wbl family proteins, especially Whi B3 proteins. It mainly includes the following five aspects: 1. Systematic analysis of regulatory proteins of Mycobacterium tuberculosis. Listeri-based monocytogenes Mog R protein contains N-terminal DNA binding domain and C-terminal dimerization domain. Mog R1-220 can bind DNA, but can not inhibit transcription. Based on this, we constructed an expression plasmid fusing Mog R with regulatory protein. The reporter plasmid was obtained by introducing the reporter gene lac Z into the Ptac promoter. After the construction of the system, the regulatory proteins of Mtb were systematically identified. 109 of the 154 regulatory proteins were probably homologous dimer proteins and 32 were probably monomer proteins. Among these monomer proteins, Wbl family protein Whi B1-Whi B7 was included. The interaction between Wbl family proteins and_A was detected by bacterial two-hybrid system. It was found that the R515 site of_A affected the interaction with Whi B3, Whi B4, Whi B6 and Whi B7, but could not affect the interaction with Whi B1 and Whi B2. Whi B3 played an important role in MTB, and Whi B3 was the research object. Further studies on the interaction were carried out. The K508, P517 and S518 loci of_A and E81 and E83 of Whi B3 were detected by bacterial two-hybrid system and protein co-purification analysis. It was found that E81 and E83 of_A and Whi B3 significantly affected their interaction. 3. Mtb Whi B3 overexpressed Mtb in Mycobacterium smegmatis (Ms). Whi B3 of M_s wild type and whi B3 overexpressed M_tb showed growth retardation, cell lengthening and cell sedimentation acceleration, while in A R515 mutant strain, these overexpression effects disappeared. Ribose was selected. Body-related genes Msrps F and Msrps J were studied. It was found that the expression of Whi B3 did not affect the transcription of these two genes, and no significant transcriptional changes were detected in MS whi B3, indicating that overexpression of Mtb Whi B3 produced different regulatory effects at different stages. 4. Whi B3 regulated the expression of MMAR_3796-MMAR_3. Transcription of 800 operons. After mutation of M. marinum (Mm) whi B3, the production of pigment was affected, and the lysis of erythrocytes and the uptake of ethidium bromide (EB) were affected. The production of pigment was related to lipid synthesis. Further detection of Mm lipid synthesis-related gene transcription in WT and whi B3 revealed that the transcription of MMAR_ 96 was significantly regulated. Transcription analysis of the whole operon gene revealed that all five genes were regulated by Whi B3. 5. Transcription activation of whi B3 was regulated by Whi B3 in acidic environment. P Ho P was phosphorylated to regulate the transcription of whi B3 under acidic conditions. Different replication experiments were carried out against the background of two mutants of Mycobacterium smegmatis Pho PR and whi B3. It was found that the activation of whi B3 under acidic conditions required phosphorylation of P Ho R and PHO P.
【学位授予单位】：中国科学院研究生院(武汉病毒研究所)
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R378.911;Q78
本文编号：2234417
[Abstract]:Mycobacterium tuberculosis (Mtb) can activate infection or latent infection according to the changes of external environment during infection. In the process of infection control, the regulation of various physiological processes of Mtb is necessary, in which transcriptional regulation plays an indispensable role in the regulation of physiological processes, and transcriptional regulation of eggs. White participates in the transcriptional regulation process through different regulatory mechanisms. To explore the regulatory mechanisms of Mtb transcriptional regulatory proteins, this paper focuses on the function and regulatory mechanisms of Wbl family proteins, especially Whi B3 proteins. It mainly includes the following five aspects: 1. Systematic analysis of regulatory proteins of Mycobacterium tuberculosis. Listeri-based monocytogenes Mog R protein contains N-terminal DNA binding domain and C-terminal dimerization domain. Mog R1-220 can bind DNA, but can not inhibit transcription. Based on this, we constructed an expression plasmid fusing Mog R with regulatory protein. The reporter plasmid was obtained by introducing the reporter gene lac Z into the Ptac promoter. After the construction of the system, the regulatory proteins of Mtb were systematically identified. 109 of the 154 regulatory proteins were probably homologous dimer proteins and 32 were probably monomer proteins. Among these monomer proteins, Wbl family protein Whi B1-Whi B7 was included. The interaction between Wbl family proteins and_A was detected by bacterial two-hybrid system. It was found that the R515 site of_A affected the interaction with Whi B3, Whi B4, Whi B6 and Whi B7, but could not affect the interaction with Whi B1 and Whi B2. Whi B3 played an important role in MTB, and Whi B3 was the research object. Further studies on the interaction were carried out. The K508, P517 and S518 loci of_A and E81 and E83 of Whi B3 were detected by bacterial two-hybrid system and protein co-purification analysis. It was found that E81 and E83 of_A and Whi B3 significantly affected their interaction. 3. Mtb Whi B3 overexpressed Mtb in Mycobacterium smegmatis (Ms). Whi B3 of M_s wild type and whi B3 overexpressed M_tb showed growth retardation, cell lengthening and cell sedimentation acceleration, while in A R515 mutant strain, these overexpression effects disappeared. Ribose was selected. Body-related genes Msrps F and Msrps J were studied. It was found that the expression of Whi B3 did not affect the transcription of these two genes, and no significant transcriptional changes were detected in MS whi B3, indicating that overexpression of Mtb Whi B3 produced different regulatory effects at different stages. 4. Whi B3 regulated the expression of MMAR_3796-MMAR_3. Transcription of 800 operons. After mutation of M. marinum (Mm) whi B3, the production of pigment was affected, and the lysis of erythrocytes and the uptake of ethidium bromide (EB) were affected. The production of pigment was related to lipid synthesis. Further detection of Mm lipid synthesis-related gene transcription in WT and whi B3 revealed that the transcription of MMAR_ 96 was significantly regulated. Transcription analysis of the whole operon gene revealed that all five genes were regulated by Whi B3. 5. Transcription activation of whi B3 was regulated by Whi B3 in acidic environment. P Ho P was phosphorylated to regulate the transcription of whi B3 under acidic conditions. Different replication experiments were carried out against the background of two mutants of Mycobacterium smegmatis Pho PR and whi B3. It was found that the activation of whi B3 under acidic conditions required phosphorylation of P Ho R and PHO P.
【学位授予单位】：中国科学院研究生院(武汉病毒研究所)
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R378.911;Q78
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1 冯立鹏;结核分枝杆菌Wbl家族蛋白分析和WhiB3功能研究[D];中国科学院研究生院(武汉病毒研究所);2016年
，本文编号：2234417
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