結(jié)核分枝桿菌Wbl家族蛋白分析和WhiB3功能研究
發(fā)布時(shí)間：2018-09-10 12:18
【摘要】：結(jié)核分枝桿菌(Mycobacterium tuberculosis,Mtb)在感染過程中可以根據(jù)外界環(huán)境變化選擇進(jìn)行激活感染或潛伏感染。在調(diào)控感染的過程中,Mtb各個(gè)生理過程的調(diào)控是必需的,其中轉(zhuǎn)錄調(diào)控在生理過程調(diào)控中發(fā)揮不可或缺的作用,而轉(zhuǎn)錄調(diào)控蛋白通過不同調(diào)控機(jī)制參與到轉(zhuǎn)錄調(diào)控過程。為探討Mtb轉(zhuǎn)錄調(diào)控蛋白的調(diào)控機(jī)制,本論文圍繞Wbl家族蛋白,特別是Whi B3的功能與調(diào)控機(jī)制開展研究,主要包括以下5方面的內(nèi)容:1.結(jié)核分枝桿菌調(diào)控蛋白的系統(tǒng)分析�；趩卧隼钏固鼐�(Listeria monocytogenes)抑制蛋白Mog R的抑制機(jī)理,構(gòu)建了單體蛋白檢測(cè)系統(tǒng)。Mog R蛋白包含N端的DNA結(jié)合結(jié)構(gòu)域和C端的二聚化結(jié)構(gòu)域。其中Mog R1-220能結(jié)合DNA,但不能抑制轉(zhuǎn)錄,基于此我們構(gòu)建了將Mog R與調(diào)控蛋白融合的表達(dá)質(zhì)粒。同時(shí)通過PCR將Mog R結(jié)合基序引入報(bào)告基因lac Z前的Ptac啟動(dòng)子中,得到報(bào)告質(zhì)粒。系統(tǒng)構(gòu)建后并對(duì)Mtb的調(diào)控蛋白進(jìn)行了系統(tǒng)鑒定分析。154個(gè)調(diào)控蛋白中有109個(gè)可能是同源二聚體蛋白,32個(gè)可能為單體蛋白。在這些單體蛋白中,包括Wbl家族蛋白Whi B1-Whi B7。采用細(xì)菌雙雜交系統(tǒng)檢測(cè)Whi B1-Whi B7的自身相互作用,結(jié)果證明它們可能作為單體蛋白發(fā)揮調(diào)控功能。2.Wbl家族蛋白與σA相互作用的檢測(cè)。通過蛋白共純化、細(xì)菌雙雜交和分枝桿菌蛋白互補(bǔ)等方法檢測(cè)Wbl家族蛋白Whi B1-Whi B7與σA的相互作用,發(fā)現(xiàn)除Whi B5外都可以與σA相互結(jié)合。經(jīng)過細(xì)菌雙雜交系統(tǒng)和分枝桿菌蛋白互補(bǔ)系統(tǒng)檢測(cè),發(fā)現(xiàn)σA的R515位點(diǎn)影響與Whi B3、Whi B4、Whi B6和Whi B7的相互作用,而不能影響與Whi B1和Whi B2的相互作用。Whi B3在Mtb中發(fā)揮重要功能,以Whi B3為研究對(duì)象進(jìn)行相互作用的進(jìn)一步研究。采用細(xì)菌雙雜交系統(tǒng)和蛋白共純化分析,利用一系列突變體進(jìn)行檢測(cè),發(fā)現(xiàn)σA的K508、P517和S518位點(diǎn)以及Whi B3的E81和E83會(huì)顯著影響它們的相互作用。3.Mtb Whi B3的生理功能。在恥垢分枝桿菌(M.smegmatis,Ms)中過表達(dá)Mtb的Whi B3,不同生長期和缺氧條件取樣,檢測(cè)發(fā)現(xiàn)whi B3的轉(zhuǎn)錄在穩(wěn)定期和缺氧時(shí)被誘導(dǎo)。在Ms野生型和Δwhi B3中誘導(dǎo)過表達(dá)Mtb的Whi B3后,表現(xiàn)為生長滯后、菌體加長和菌體沉降加快;而在σA的R515突變體菌株中,這些過表達(dá)效應(yīng)消失。選取核糖體相關(guān)基因Msrps F和Msrps J為靶標(biāo)進(jìn)行研究,發(fā)現(xiàn)在穩(wěn)定期添加誘導(dǎo)劑誘導(dǎo)Mtb的Whi B3表達(dá)沒有影響這兩個(gè)基因的轉(zhuǎn)錄,同時(shí)在MsΔwhi B3中檢測(cè)不到明顯的轉(zhuǎn)錄變化,表明不同時(shí)期過表達(dá)Mtb Whi B3產(chǎn)生不同的調(diào)控效應(yīng)。4.Whi B3調(diào)控MMAR_3796-MMAR_3800操縱子的轉(zhuǎn)錄。在海分枝桿菌(M.marinum,Mm)突變whi B3后影響菌體色素的產(chǎn)生、對(duì)紅細(xì)胞的裂解和溴化乙錠(EB)攝取,其中色素的產(chǎn)生與脂類合成相關(guān)。進(jìn)一步在WT和Δwhi B3中檢測(cè)Mm脂類合成相關(guān)基因的轉(zhuǎn)錄,發(fā)現(xiàn)MMAR_3796的轉(zhuǎn)錄受到明顯調(diào)控。對(duì)整個(gè)操縱子基因的轉(zhuǎn)錄進(jìn)行分析,發(fā)現(xiàn)該操縱子的5個(gè)基因均受到Whi B3的調(diào)控。5.酸性環(huán)境下whi B3轉(zhuǎn)錄激活調(diào)控機(jī)制。通過對(duì)whi B3的啟動(dòng)子(whi B3p)分析和體外EMSA實(shí)驗(yàn),確定Pho P可以特異性結(jié)合whi B3p。回復(fù)Mm的pho P和whi B3雙基因突變體,發(fā)現(xiàn)在酸性條件下Pho P被磷酸化后調(diào)控whi B3的轉(zhuǎn)錄。以恥垢分枝桿菌pho PR和whi B3雙突變體為背景進(jìn)行不同回復(fù)實(shí)驗(yàn),發(fā)現(xiàn)酸性條件下whi B3的轉(zhuǎn)錄激活需要Pho R和Pho P的磷酸化過程。
[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.
【學(xué)位授予單位】：中國科學(xué)院研究生院(武漢病毒研究所)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R378.911;Q78
本文編號(hào)：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.
【學(xué)位授予單位】：中國科學(xué)院研究生院(武漢病毒研究所)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R378.911;Q78
【相似文獻(xiàn)】
相關(guān)博士學(xué)位論文 前1條
1 馮立鵬;結(jié)核分枝桿菌Wbl家族蛋白分析和WhiB3功能研究[D];中國科學(xué)院研究生院(武漢病毒研究所);2016年
，本文編號(hào)：2234417
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