分枝杆菌噬菌体的系统生物学研究

发布时间：2018-05-18 21:57

本文选题：分枝杆菌噬菌体 + 全基因组测序　；参考：《西南大学》2014年博士论文

【摘要】：分枝杆菌属是隶属于放线菌目的一类细菌,两种常见的致病菌：结核分枝杆菌(Mycobacterium tuberculosi)以及麻风分枝杆菌(M. leprae)都是分枝杆菌属内的一员。自从1981年卫生部提出20世纪末基本消灭麻风的目标后,目前由麻风分枝杆菌引起的麻风病在我国已得到有效的控制。而由结核分枝杆菌所引起的结核病则因为日益严重的耐药性、与HIV的共感染以及卡介苗的不稳定性等呈现重新蔓延的趋势。控制结核病已成为科研及医疗工作者所面临的巨大任务。分枝杆菌噬菌体是一类可感染分枝杆菌属细菌的噬菌体,对其研究起始于1950s。随着研究的深入,分枝杆菌噬菌体己成为控制结核病的重要工具,比如：分枝杆菌噬菌体以及其衍生物已逐渐被开发为基础研究中分枝杆菌的遗传操作载体和临床上(耐药)结核菌的有效诊断工具；部分噬菌体组分也被研究,其中的一些被认为具有开发为新一代抗结核药物的潜力。但令人遗憾的是,现在基于分枝杆菌噬菌体开发出的结核菌分子操作载体以及结核病诊断工具的专利权都集中在欧美。因此为了对我国开发具有自主知识产权的结核病控制工具提供源头上的创新,分离中国特有的分枝杆菌噬菌体并对其进行全基因组测序势在必行。另外,研究分枝杆菌噬菌体与宿主菌相互作用的机理、一些噬菌体宿主关闭蛋白的功能、分枝杆菌原噬菌体多样性及其与结核菌致病性的关系都为利用噬菌体控制结核病提供了基础。基于以上几点,本论文主要从以下几个部分进行研究：首先我们分离得到了一株新的分枝杆菌噬菌体,并将其命名为SWU1。这株噬菌体感染耻垢分枝杆菌(M. smegmatis mc2155)后形成周围有晕环的牛眼状噬菌斑,电镜观察发现其为典型的蝌蚪状。得到其全基因组序列后,分析发现其属于分枝杆菌噬菌体A2群体。针对SWU1与L5(A2群体典型噬菌体)表型的不同,进行了遗传学上的解释,分析认为SWU1中的gp39可能具有多糖解聚酶的功能。在分离分枝杆菌噬菌体SWU1的过程中,我们发现现有的分枝杆菌噬菌体分类鉴定方法具有较大的缺点,如必须进行全基因组测序、花费较大等。为了解决这一难题,我们开发了一种新的基于保守基因的分枝杆菌噬菌体鉴定新方法。利用68个可获取全基因组的分枝杆菌噬菌体,我们首先获得分枝杆菌噬菌体的核心基因组。之后,我们分析了其中的核心基因,发现A类核心基因非常保守,最适合被用来鉴定分枝杆菌噬菌体。最后,针对A类核心基因的保守位点,我们设计了针对不同分枝杆菌噬菌体簇的引物对。其中针对分枝杆菌噬菌体A2簇的引物被验证是可行的。作为细菌的病毒,噬菌体在进化过程中获得了一些可控制宿主细菌正常生理代谢的蛋白质。而这些蛋白质本身或其作用位点、作用方式是开发新型抗生素的模型。为了更好的了解分枝杆菌噬菌体是如何侵染、控制其宿主菌的,我们首次利用RNA-seq技术研究了分枝杆菌噬菌体SWUl与其宿主耻垢分枝杆菌的互作。数据分析显示,宿主会有一些防御噬菌体的反应,比如,诱导信号传导系统从全局应对噬菌体的感染,上调细胞壁合成基因试图修复被噬菌体破坏的细胞壁。在此过程,噬菌体也劫持了菌体的蛋白质、DNA及RNA的合成系统,破坏了菌体的铁摄取系统以帮助自身的繁殖。其中破坏分枝杆菌的铁摄取系统也许是开发新型抗结核药物的新理念。依靠RNA-seq技术从全局上了解到宿主菌与噬菌体SWU1的相互作用后,我们更想知道的是单个蛋白质在噬菌体侵染宿主菌过程中所发挥的功能。其中分枝杆菌噬菌体D29的gp34.1吸引了我们。这一蛋白存在一个DUF2637超家族结构域。在耻垢分枝杆菌内表达此蛋白质后,发现其菌落形态改变,且重组菌获得耐受噬菌体TM4的表型。噬菌体仍然可以正常的吸附并将DNA注入进重组菌体内。进一步研究显示其可抑制大肠杆菌的生长,是一个新发现的宿主关闭蛋白。通过蛋白截短实验探索到D29gp34.1具有宿主毒性的核心作用区域为第26个氨基酸到第100个氨基酸区段的肽段。我们认为这一多肽片段具有开发为新型抗生素骨架的潜力。有研究表明分枝杆菌基因组内存在大量的原噬菌体,为了更好的了解噬菌体与分枝杆菌致病性的关系,我们对分枝杆菌基因组内的原噬菌体进行了系统的挖掘、比较并探讨了其与分枝杆菌致病性的关系。总共搜索到了33个分枝杆菌原噬菌体,其中11个是之前未报道的原噬菌体。通过对全部33个原噬菌体的全基因组的比较：首次发现位于M.massiliense Strain M172基因组上的全长原噬菌体phil72_2可被归为分枝杆菌噬菌体A簇；另外,发现了一组缺陷原噬菌体phiMmcs_1(phiMkms_1)、phiBN44_1以及phiMCAN_1具有同源性,可以被归为同一类。
[Abstract]:Mycobacterium tuberculosis is a kind of bacterium belonging to Actinomyces , two common pathogenic bacteria : Mycobacterium tuberculosis and M . leprae are a member of the genus Mycobacterium .

The mycobacterial phage is a kind of phage that can infect the bacterium of Mycobacterium , which starts from the research . With the development of the research , the mycobacterium phage has become an important tool for controlling tuberculosis , such as Mycobacterium tuberculosis phage and its derivative has been developed into the genetic operation carrier of mycobacterium in the foundation study and the effective diagnostic tool for clinical ( resistant ) mycobacterium tuberculosis ;
Some of the bacteriophage components are also studied , some of which are thought to have the potential to develop a new generation of antitubercular drugs . However , it is regrettable that the Mycobacterium tuberculosis molecular manipulation vector and the patent right of tuberculosis diagnostic tools are now concentrated in Europe and America . In addition , it is necessary to study the mechanism of the development of tuberculosis control tools with independent intellectual property rights .

A new phage of Mycobacterium smegmatis mc2155 was isolated and named SWU1 . The phage infected with Mycobacterium smegmatis mc2155 was infected with M . smegmatis mc2155 . It was found that it was a typical tadpole . After the whole genome sequence was obtained , it was found that it belonged to Mycobacterium tuberculosis phage A2 population . In this paper , genetic explanation was made for the phenotype of SWU1 and L5 ( typical phage ) . It was suggested that gp39 in SWU1 might have the function of polysaccharide depolymerase .

In order to solve this problem , we have developed a new method for the identification of Mycobacterium tuberculosis phage . In order to solve this problem , we have developed a new method for the identification of mycobacterial phage based on conserved genes .

In order to better understand how to infect and control the host bacteria of the host bacteria , we first use RNA - seq technology to study the interaction between the mycobacterial phage SWUl and its host smegmatis . In this process , the phage also holds the protein , DNA and RNA synthesis system of the bacteria , destroying the iron intake system of the bacteria to help its own reproduction .

From the global perspective on the interaction of host bacteria with phage SWU1 , we would like to know the function of a single protein in the process of phage infection host bacteria . The protein has a DUF2637 superfamily domain . The protein has a DUF2637 superfamily domain .

A large number of prophage were found in the genome of Mycobacterium tuberculosis . In order to better understand the relationship between the pathogenicity of phage and Mycobacterium tuberculosis , we compared and discussed the relationship between phage and mycobacterial pathogenicity . In total , 33 Mycobacterium prophage were searched , 11 of them were previously unreported . By comparison of the whole genome of all 33 prophage : the first finding that the full - length prophage phil72 _ 2 located on the genome of M . massiliense Strain M172 can be classified as Mycobacterium tuberculosis phage A cluster ;
In addition , it has been found that phiMkms _ 1 ( phiMkms _ 1 ) , philist44 _ 1 and phiMCAN _ 1 have homology and can be classified into the same class .
【学位授予单位】：西南大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R378.911;Q93

【参考文献】