海分枝杆菌致病基因的分离鉴定及功能研究

发布时间：2018-04-01 21:09

本文选题：结核分枝杆菌　切入点：海分枝杆菌　出处：《复旦大学》2010年博士论文

【摘要】：由结核分枝杆菌引起的结核病至今仍然是一个很重要的全球性健康问题。全球每年结核病新发病例约为1000万,而由结核导致的死亡每年高达约200万人。这一状况随着结核与HIV共感染,以及结核耐多药菌株和超级耐药菌株的出现而进一步恶化。鉴于此,寻找开发新的抗结核药物显得尤为迫切,而此过程需要对结核分枝杆菌的生理致病性、以及在宿主细胞中的生活习性做一个系统的理解。海分枝杆菌的天然宿主为鱼类和两栖类,在遗传上与结核分枝杆菌极为相近,很多研究显示海分枝杆菌与结核分枝杆菌享有很多共同的毒力因子及致病机制,是研究结核分枝杆菌毒力、揭示结核发病机制很好的模型。本研究以海分枝杆菌为模型,研究结核分枝杆菌致病基因功能及结核分枝杆菌的发病机制。研究的总策略：在海分枝杆菌中建立转座子随机插入突变库,并以菌落表型变化为筛选标准,筛选细菌细胞壁成份发生变化的突变菌株；进一步运用索状结构表型以及斑马鱼毒力实验模型筛选毒力相关基因突变；以分子生物学、细胞、生化等研究手段,研究新鉴定的致病相关基因的生物学功能。利用MycoMarT7转座子随机插入突变技术,我们在海分枝杆菌中建立了个库容量约为104的转座子随机插入突变文库；通过菌落形态变化,共筛选出66个细菌菌落发生改变的突变菌株；运用抗性标记挽救法成功鉴定出其中53个突变菌株的转座子插入位点,涉及48个基因；其中,在其他细菌中已有研究报道跟毒力相关的同源基因15个,占所筛选的总基因的31.25%。充分说明从海分枝杆菌菌落表型变化筛选与毒力可能相关基因策略的可行性。同时为能够更有效筛选毒力直接相关基因,我们建立了斑马鱼毒力感染模型,用以筛选对斑马鱼致病性发生减弱的突变菌株；本研究为海分枝杆菌致病基因的筛选提供了新策略；同时为也为接下来研究海分枝杆菌致病机制提供了候选基因。分支杆菌的细胞表面结构复杂、成分多样,在细菌抵制外界不良环境以及在细菌操纵宿主免疫系统过程中发挥重要作用。索状结构表型是结核分枝杆菌复合群中致病分支杆菌特有表型。在已经建立的突变库中运用索状结构表型,我们成功鉴定出9株索状结构表型消失的突变,并且其中7株都为脂质PDIMs的合成通路中基因的突变(fadD26, ppsA, ppsB, ppsD, ppsE, mas, fadD28)。PDIMs为分支杆菌细胞壁重要的脂质成分,在结核分枝杆菌中有报道跟毒力相关,但是具体的致病机制仍不清楚。我们对鉴定出的PDIMs缺失突变株深入研究发现,PDIMs为海分枝杆菌重要的致病因子,该脂质对海分枝杆菌在斑马鱼中的增殖以及海分枝杆菌逃逸宿主免疫系统过程中都发挥重要作用。另外鉴定出两个索状结构表型相关的新基因PPE38和mmaA3。PPE38为PPE蛋白家族成员,功能未知；mmaA3预测编码甲基氧胆酸酯合酶,具体功能也尚未报道。运用斑马鱼感染模型我们发现,这两个索状结构缺失的突变菌株毒力较野生型没有发生显著性改变,即索状结构的有无跟菌株毒力的强弱并没有必然联系。这一发现将对传统的认为索状结构是有毒分支杆菌特征表型这一观点提出挑战。生物素为所有生命体所必需,在脂肪酸合成、氨基酸代谢、以及碳水化合物的代谢过程中的羧化反应中起重要作用。大多数的微生物、植物以及真菌都能够自身合成生物素,而哺乳动物及人类需要从食物中或者大肠中的共生菌中摄取。生物素由pimeloyl-CoA经bioF编码的KAPA合成酶、bioA编码的DAPA合成酶、bioD编码的去生物素合成酶、以及bioB编码的生物素合成酶四步酶促反应合成,且这一通路在格兰阳性菌和革兰阴性菌中都很保守。分支杆菌中同样存在生物素合成相关基因,耻垢分支杆菌中生物信息分析存在bioA基因,且该基因的突变可造成耻垢分支杆菌在平台期生长发生变化：另外在结核分枝杆菌中体外对Rv1569基因编码的KAPA合成酶以及Rv1568基因编码的DAPA合成酶的生化活性也有研究；但至今尚未在致病分支杆菌中直接鉴定生物素合成通路基因,生物素合成在分支杆菌感染过程中发挥何种功能也未有报道。我们在已建立的突变库中筛选得到一生物素合成相关基因的突变菌株,为MMAR_2770基因的插入失活突变。MMAR2770基因预测编码一个短链脱氢酶家族成员酶,具体功能尚未有研究；我们的研究表明该基因的突变使得海分枝杆菌体外生长呈生物素依赖表型,在小鼠巨噬细胞以及斑马鱼中的毒力减弱；并且该基因在结核分枝杆菌中的同源基因Rv1882c能够回复该基因所产生的突变表型,表明该基因在分支杆菌中功能保守。同时人类基因组中不存在MMAR2770基因的同源基因,使其成为一个很好的潜在的抗结核药物作用靶标。
[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 .

【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R378.911

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