本文選題：結(jié)核分枝桿菌　切入點(diǎn)：海分枝桿菌　出處：《復(fù)旦大學(xué)》2010年博士論文

【摘要】：由結(jié)核分枝桿菌引起的結(jié)核病至今仍然是一個(gè)很重要的全球性健康問題。全球每年結(jié)核病新發(fā)病例約為1000萬,而由結(jié)核導(dǎo)致的死亡每年高達(dá)約200萬人。這一狀況隨著結(jié)核與HIV共感染,以及結(jié)核耐多藥菌株和超級(jí)耐藥菌株的出現(xiàn)而進(jìn)一步惡化。鑒于此,尋找開發(fā)新的抗結(jié)核藥物顯得尤為迫切,而此過程需要對(duì)結(jié)核分枝桿菌的生理致病性、以及在宿主細(xì)胞中的生活習(xí)性做一個(gè)系統(tǒng)的理解。海分枝桿菌的天然宿主為魚類和兩棲類,在遺傳上與結(jié)核分枝桿菌極為相近,很多研究顯示海分枝桿菌與結(jié)核分枝桿菌享有很多共同的毒力因子及致病機(jī)制,是研究結(jié)核分枝桿菌毒力、揭示結(jié)核發(fā)病機(jī)制很好的模型。 本研究以海分枝桿菌為模型,研究結(jié)核分枝桿菌致病基因功能及結(jié)核分枝桿菌的發(fā)病機(jī)制。研究的總策略：在海分枝桿菌中建立轉(zhuǎn)座子隨機(jī)插入突變庫,并以菌落表型變化為篩選標(biāo)準(zhǔn),篩選細(xì)菌細(xì)胞壁成份發(fā)生變化的突變菌株；進(jìn)一步運(yùn)用索狀結(jié)構(gòu)表型以及斑馬魚毒力實(shí)驗(yàn)?zāi)Ｐ秃Y選毒力相關(guān)基因突變；以分子生物學(xué)、細(xì)胞、生化等研究手段,研究新鑒定的致病相關(guān)基因的生物學(xué)功能。 利用MycoMarT7轉(zhuǎn)座子隨機(jī)插入突變技術(shù),我們?cè)诤７种U菌中建立了個(gè)庫容量約為104的轉(zhuǎn)座子隨機(jī)插入突變文庫；通過菌落形態(tài)變化,共篩選出66個(gè)細(xì)菌菌落發(fā)生改變的突變菌株；運(yùn)用抗性標(biāo)記挽救法成功鑒定出其中53個(gè)突變菌株的轉(zhuǎn)座子插入位點(diǎn),涉及48個(gè)基因；其中,在其他細(xì)菌中已有研究報(bào)道跟毒力相關(guān)的同源基因15個(gè),占所篩選的總基因的31.25%。充分說明從海分枝桿菌菌落表型變化篩選與毒力可能相關(guān)基因策略的可行性。同時(shí)為能夠更有效篩選毒力直接相關(guān)基因,我們建立了斑馬魚毒力感染模型,用以篩選對(duì)斑馬魚致病性發(fā)生減弱的突變菌株；本研究為海分枝桿菌致病基因的篩選提供了新策略；同時(shí)為也為接下來研究海分枝桿菌致病機(jī)制提供了候選基因。 分支桿菌的細(xì)胞表面結(jié)構(gòu)復(fù)雜、成分多樣,在細(xì)菌抵制外界不良環(huán)境以及在細(xì)菌操縱宿主免疫系統(tǒng)過程中發(fā)揮重要作用。索狀結(jié)構(gòu)表型是結(jié)核分枝桿菌復(fù)合群中致病分支桿菌特有表型。在已經(jīng)建立的突變庫中運(yùn)用索狀結(jié)構(gòu)表型,我們成功鑒定出9株索狀結(jié)構(gòu)表型消失的突變,并且其中7株都為脂質(zhì)PDIMs的合成通路中基因的突變(fadD26, ppsA, ppsB, ppsD, ppsE, mas, fadD28)。PDIMs為分支桿菌細(xì)胞壁重要的脂質(zhì)成分,在結(jié)核分枝桿菌中有報(bào)道跟毒力相關(guān),但是具體的致病機(jī)制仍不清楚。我們對(duì)鑒定出的PDIMs缺失突變株深入研究發(fā)現(xiàn),PDIMs為海分枝桿菌重要的致病因子,該脂質(zhì)對(duì)海分枝桿菌在斑馬魚中的增殖以及海分枝桿菌逃逸宿主免疫系統(tǒng)過程中都發(fā)揮重要作用。另外鑒定出兩個(gè)索狀結(jié)構(gòu)表型相關(guān)的新基因PPE38和mmaA3。PPE38為PPE蛋白家族成員,功能未知；mmaA3預(yù)測(cè)編碼甲基氧膽酸酯合酶,具體功能也尚未報(bào)道。運(yùn)用斑馬魚感染模型我們發(fā)現(xiàn),這兩個(gè)索狀結(jié)構(gòu)缺失的突變菌株毒力較野生型沒有發(fā)生顯著性改變,即索狀結(jié)構(gòu)的有無跟菌株毒力的強(qiáng)弱并沒有必然聯(lián)系。這一發(fā)現(xiàn)將對(duì)傳統(tǒng)的認(rèn)為索狀結(jié)構(gòu)是有毒分支桿菌特征表型這一觀點(diǎn)提出挑戰(zhàn)。 生物素為所有生命體所必需,在脂肪酸合成、氨基酸代謝、以及碳水化合物的代謝過程中的羧化反應(yīng)中起重要作用。大多數(shù)的微生物、植物以及真菌都能夠自身合成生物素,而哺乳動(dòng)物及人類需要從食物中或者大腸中的共生菌中攝取。生物素由pimeloyl-CoA經(jīng)bioF編碼的KAPA合成酶、bioA編碼的DAPA合成酶、bioD編碼的去生物素合成酶、以及bioB編碼的生物素合成酶四步酶促反應(yīng)合成,且這一通路在格蘭陽性菌和革蘭陰性菌中都很保守。分支桿菌中同樣存在生物素合成相關(guān)基因,恥垢分支桿菌中生物信息分析存在bioA基因,且該基因的突變可造成恥垢分支桿菌在平臺(tái)期生長發(fā)生變化：另外在結(jié)核分枝桿菌中體外對(duì)Rv1569基因編碼的KAPA合成酶以及Rv1568基因編碼的DAPA合成酶的生化活性也有研究；但至今尚未在致病分支桿菌中直接鑒定生物素合成通路基因,生物素合成在分支桿菌感染過程中發(fā)揮何種功能也未有報(bào)道。我們?cè)谝呀⒌耐蛔儙熘泻Y選得到一生物素合成相關(guān)基因的突變菌株,為MMAR_2770基因的插入失活突變。MMAR2770基因預(yù)測(cè)編碼一個(gè)短鏈脫氫酶家族成員酶,具體功能尚未有研究；我們的研究表明該基因的突變使得海分枝桿菌體外生長呈生物素依賴表型,在小鼠巨噬細(xì)胞以及斑馬魚中的毒力減弱；并且該基因在結(jié)核分枝桿菌中的同源基因Rv1882c能夠回復(fù)該基因所產(chǎn)生的突變表型,表明該基因在分支桿菌中功能保守。同時(shí)人類基因組中不存在MMAR2770基因的同源基因,使其成為一個(gè)很好的潛在的抗結(jié)核藥物作用靶標(biāo)。
[Abstract]:Tuberculosis is still a very important global health problem , which is caused by Mycobacterium tuberculosis . The annual rate of tuberculosis in the world is about 10 million , and the mortality caused by tuberculosis is about 2 million per year . In view of this , it is especially urgent to find new anti - TB drugs . In view of this , it is very urgent to find new anti - TB drugs . Many studies show that mycobacterium tuberculosis and Mycobacterium tuberculosis have many common virulence factors and pathogenic mechanisms , and are a good model to study the virulence of Mycobacterium tuberculosis and reveal the pathogenesis of tuberculosis .

In this study , the pathogenic gene function of Mycobacterium tuberculosis and the pathogenesis of Mycobacterium tuberculosis were studied by means of Mycobacterium tuberculosis .
and screening the virulence - related gene mutation by using the phenotype of the rope - like structure and the zebra fish virulence test model ;
The biological function of newly identified pathogenic - related genes was studied by means of molecular biology , cell , biochemistry and so on .

Using the MycoMarT7 transposon random insertion mutation technique , we established a transposon random insertion mutation library with a library capacity of about 104 in Mycobacterium avium ;
The mutant strains were screened out of 66 bacterial colonies by colony morphology .
The transposon insertion sites of 53 mutant strains were successfully identified by the method of resistance marker salvage , involving 48 genes .
Among other bacteria , 15 of the homologous genes related to virulence were reported , accounting for 31.25 % of the screened total genes .
This study provides a new strategy for the selection of the pathogenic genes of Mycobacterium species .
At the same time , candidate genes were also provided for the next study of the pathogenic mechanism of M . M . M .

The cell surface of Mycobacterium tuberculosis has a complex structure and various components , plays an important role in bacteria resistance to external environment and plays an important role in the process of bacterial manipulation of host immune system . PDIMs is an important lipid component of the cell wall of Mycobacterium tuberculosis , but the specific pathogenic mechanism is not clear . We have found that PDIMs plays an important role in the proliferation of M .
mmaA3 predicted that there was no significant change in the virulence of the mutant strains , i.e . the presence or absence of the sordid structure and the virulence of the strain , which would be a challenge to the traditional view of the characteristic phenotype of the virulent mycobacterial species .

In addition , biotin biosynthesis - related gene , bioA - encoded debiotin synthetase , bioD - encoded debiotin synthetase , and bioB - encoded biotin synthetase four - step enzymatic reaction synthesis , and the mutation of the gene can lead to a change in the growth of Mycobacterium smegmatis in the platform period , and the biochemical activity of DAPA synthetase encoded by Rv1569 gene in vitro in Mycobacterium tuberculosis is also studied ;
However , it has not been reported that the biotin synthesis pathway gene has not been directly identified in the pathogenic branch bacilli , and the biotin synthesis has not been reported in the process of mycobacterial infection . We screened a mutant strain of biotin synthesis related gene in the established mutant library , which is an insertion inactivation mutation of the MMAR _ 2770 gene . The MMAR2770 gene predicts a short - chain dehydrogenase family member enzyme , and the specific function has not been studied ;
Our study shows that the mutation of the gene causes the growth of mycobacterium in vitro to be biotin - dependent phenotype , and the virulence of mouse macrophages and zebra fish weakens ;
and the homologous gene Rv182c of the gene in the mycobacterium tuberculosis can reply to the mutant phenotype generated by the gene , indicating that the gene is functionally conserved in the mycobacterium , and meanwhile , the homologous gene of the MMAR2770 gene is not present in the human genome , so that it is a good potential anti - tuberculosis drug action target .

【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2010
【分類號(hào)】：R378.911

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 莊玉輝;張寅;韓元華;劉志恒;阮繼生;;抗酸分枝桿菌和相關(guān)菌全細(xì)胞枝菌酸甲基酯的薄層分析[J];微生物學(xué)報(bào);1989年01期

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本文編號(hào)：1697290