两株肉毒梭菌（A str.230611，F str.230613）全基因组测序分析及pH环境因素对毒素基因表达的影响、调

发布时间：2018-01-15 03:23

本文关键词：两株肉毒梭菌（A str.230611，，F str.230613）全基因组测序分析及pH环境因素对毒素基因表达的影响、调控研究　出处：《中国人民解放军军事医学科学院》2011年硕士论文　论文类型：学位论文

【摘要】：肉毒梭菌(C.botulinum)是一组能产生肉毒毒素的细菌的统称。它们是一群专性厌氧的,短杆状革兰氏阳性菌。这些细菌属于梭菌属中不同的种,并且分为4个群。近年来越来越多的研究证明肉毒梭菌可以通过肠道感染致病,但是对于其粘附,侵入并定殖肠道的致病机理了解较少。肉毒毒素是肉毒梭菌致病性的重要方面。肉毒毒素的编码基因存在于一个15kb大小的毒素基因簇上。目前对于这个毒素基因簇的进化来源,不同型毒素基因簇的插入位点差异及插入规律规律,毒素基因簇结构多样性产生机制的认识比较少。为了解决以上问题,本研究测定了A型(230611)和F型(230613)肉毒梭菌全基因组序列,并且对基因组结构、基因组稳定性、肉毒梭菌进化分类、肉毒梭菌肠道感染相关基因、毒素基因簇序列及排列、毒素基因簇重组事件等进行分析和比较。肉毒梭菌肠道致病是近年来发现的一个条件性致病现象,对于其在人体消化道不同环境中的适应性及毒素基因表达调控研究较少。肉毒梭菌通过人体消化道各器官时会经历酸性、中性和碱性环境。细菌在产毒方面对pH值的改变会作出相应变化,调控自身基因表达量以适应环境并保持致病性。目前认为毒素基因簇中botR基因是毒素基因表达的调控基因,但是对于在细菌内部实际生理状态下botR基因对毒素基因bont的调控作用及转录量变化关系还缺乏系统的研究。为了深入了解肉毒梭菌对人体消化道不同pH环境的适应性,毒素基因表达的变化及调控基因botR对毒素基因bont的调控作用,我们挑选毒力较强的A型(230611)肉毒梭菌在不同pH环境下培养,测定pH值对细菌生长、毒素基因和调控基因转录量变化及相关性、毒素蛋白分泌量的差别,从而探索毒素基因表达的调控机制。第一部分A(230611)、F(230613)型肉毒梭菌全基因组序列测定、进化分类、基因组稳定性及致病基因分析我们使用第二代高通量测序技术(Roche 454 GS)测定了A型(230611)和F型(230613)肉毒梭菌全基因组序列。测序深度分别为全基因组的13.7倍和10.1倍,覆盖率均为99%以上。根据参考序列设计引物测定缺口序列,得到基因组完整的序列。使用16S rRNA聚类法对已完成全基因组测序的肉毒梭菌菌株进行进化分类,结果显示对第一群(Group I)内部菌株的聚类结果可信度较低。我们采用类似MLST的方法对相同菌株进行聚类,聚类结果能正确反映菌株的进化关系,并且能精确区分A1亚型菌株内部的进化关系。聚类结果表明,A(230611)菌株属于第一群的A1亚型菌株,在四个A1亚型菌株中最早分化;F(230613)型菌株属于第一群F型菌株,与F str. Langeland菌株进化关系最近。分析肉毒梭菌基因组中的移动元件结果显示,基因组中均不含有完整的插入序列(IS),只有6个和5个残余的插入序列(partial IS)。使用密码子偏爱性分析基因组岛(Genomic Island, GI),结果显示两个菌株不含基因组岛。以上结果表明A(230611)、F(230613)型菌株基因组比较稳定,可能与其生活周期长期处于芽孢休眠状态有关。致病基因分析表明A(230611)、F(230613)型肉毒梭菌基因组中各有9个和11个与细菌粘附和侵入相关的基因,这些基因包含粘附素(intimin)、侵袭素(invasin)、鞭毛钩相关蛋白(Flagellar hook-associated)等结构域,并且在同型的其它菌株中也存在这些基因,可能与肉毒梭菌肠道感染的粘附侵入有关,下一步可以通过实验证明这些基因的功能。与最近源物种且不产肉毒毒素的生孢梭菌基因组比对及GC含量分析表明,在进化过程中,A(230611)、F(230613)型肉毒梭菌在基因组特定位置通过水平基因转移方式,摄入一段外来的含有肉毒毒素基因簇的片段,从而获得产生肉毒毒素的能力。同时比对生孢梭菌与A1,A2、F型(230613)肉毒梭菌基因组,我们发现三个肉毒梭菌的毒素基因簇分别插入染色体不同位点。通过基因组比对,我们将9个肉毒毒素基因簇的插入位点分别定位到F型(230613)菌株基因组的三个位点。插入位点分析表明,肉毒毒素基因簇片段的插入不是随机发生的,而是有规律地插入染色体上3个位点之一。A1、B型毒素基因簇倾向于插入site 3,A2型倾向于site 1,F型(230613)倾向于site 2。各型毒素基因簇结构比较表明,非毒素基因部分具有位点特异性,而毒素基因流动性较大。我们发现site 2位点的bontF毒素基因簇与site 3位点的bontA1毒素基因簇发生了重组事件,形成新的bontA1毒素基因簇结构。这也是肉毒毒素基因簇多样性的形成机制。第二部分pH值对A型(230611)肉毒梭菌毒素基因bontA表达的影响及调控基因botR的调控作用肉毒梭菌在人体肠道感染过程中会经历酸性,中性和碱性pH环境。为了深入了解A型(230611)肉毒梭菌在产毒方面对不同pH环境的适应性,我们系统性地测定不同pH(6.0,7.0,8.0,9.0)条件下细菌不同生长时间点bontA基因的转录量,以及毒素蛋白分泌量。为了研究毒素基因簇中调控基因botR对毒素基因bontA的调控作用,我们同时测定botR基因的转录量,并且研究bontA与botR基因转录量变化的相关性。我们进一步对不同环境条件下不同生长期的细菌培养物进行转录组RNA测序,从而深入探究毒素基因表达调控机理。在转录水平,针对毒素基因bontA转录量测定结果显示:pH 7.0条件下转录量最高,酸性或碱性条件下转录量显著降低(P0.01),pH 9.0条件下转录量最低(P0.01)。提示细菌为了适应酸性和碱性环境,会在转录水平抑制毒素基因bontA的表达。在蛋白水平,针对毒素蛋白分泌量测定结果显示:pH 7.0条件下毒素蛋白分泌量最高,pH 6.0和pH 8.0条件下分泌量降低,与毒素基因转录被抑制有关。但是pH9.0条件下毒素蛋白分泌与pH7.0条件下无明显差别。推测原因为:虽然pH值9条件下bontA基因的转录受到抑制,但是转录后的翻译或转运效率增高。调控基因botR转录量测定结果显示:pH 7.0条件下botR基因转录量最高,酸性或碱性条件下转录量降低(P0.05),pH 9.0条件下转录量最低(P0.01)。botR基因与bontA基因转录量相关性分析表明:不同pH条件下botR基因和bontA基因转录量在生长过程中是正相关的,相关系数介于0.7和1.0之间。这说明botR基因是bontA基因的正调控基因,在细菌生长周期,bontA基因转录量与botR基因转录量协同变化。我们选取毒素基因转录量差别较大的pH7和pH9两个条件培养细菌,分别取指数期、稳定期和衰亡期的菌液进行转录组测序,准备通过共表达基因聚类分析来寻找与毒素基因表达相关的转录因子,顺式作用元件,以及调控RNA,如5’UTR, 3’UTR, small RNA等。
[Abstract]:Clostridium botulinum (C.botulinum) is a group of bacteria that can produce botulinum toxin. They are a group of specialized anaerobic, short rod gram positive bacteria. These bacteria belong to different species of Clostridium, and they are divided into 4 groups.
In recent years, more and more studies showed that Clostridium botulinum can through the intestinal infection, but the adhesion, invasion and colonization of the intestinal pathogenic mechanism. Little is known about the botulinum toxin is an important aspect of the pathogenicity of Clostridium botulinum. Gene encoding botulinum toxin present in the toxin gene cluster size on a 15KB. At present, the toxin gene cluster evolutionary origin, insertion and the regularity of the insertion sites of different type of toxin gene cluster, less toxin gene cluster structure diversity mechanism. In order to solve the above problems, A type were determined in this study (230611) and F (230613) whole genome sequence of Clostridium botulinum. The genome structure, genome stability, evolutionary classification of Clostridium botulinum, intestinal infection related gene of Clostridium botulinum toxin gene cluster, and sequence alignment, toxin gene cluster recombination events were analyzed and compared.
Clostridium botulinum is a conditional pathogenic intestinal pathogenic phenomenon discovered in recent years, in the human digestive tract in different environment adaptability and toxin gene expression research. Clostridium botulinum through the digestive organ of the human body will experience acidic, neutral and alkaline environment. The value of pH in the bacterial toxin producing the change will make the corresponding change, to adapt to the environment and keep the pathogenic expression of their genes. The regulation of botR toxin gene cluster genes are toxin gene expression, but the change of regulation and the relationship between the amount of transcription of BoNT gene from the lack of systematic research on bacterial botR gene internal actual physiological condition.
In order to understand the adaptability of Clostridium botulinum on human digestive tract in different pH environment, role change and expression of botR toxin gene on the expression of BoNT toxin gene, we selected strong virulent type A (230611) of Clostridium botulinum in different pH environment culture, determination of pH value on the growth of bacteria, the gene expression quantity the toxin gene and regulation of toxin protein secretion and the correlation between the quantity difference, so as to explore the regulation mechanism of toxin gene expression.
The first part of A (230611), F (230613) type of Clostridium botulinum genome sequencing, evolutionary classification, genomic stability and pathogenic gene analysis
We use the second generation high-throughput sequencing technology (Roche GS 454) A was determined (230611) and F (230613) whole genome sequence of Clostridium botulinum. The sequencing depth of whole genome were 13.7 times and 10.1 times, the coverage rate was more than 99%. According to the measured gap sequence reference sequences obtained. The complete genome sequence of Clostridium botulinum strains. The whole genome sequencing has been completed by phylogenetic classification using 16S rRNA clustering method, the results showed that the first group (Group I) clustering results of internal strains with low confidence. We use the method of MLST on the same strain clustering, clustering results can correctly reflect the phylogenetic relationship of strains, and can accurately distinguish the evolutionary relationship between internal A1 subtype strains. The clustering results showed that A (230611) strains belonging to the first group of subtype A1 strains, the earliest differentiation in the four subtype A1 strains; F (230613) strains belong to the A group of F strains are closely related to the evolution of the F str. Langeland strain.
Analysis of the moving element of Clostridium botulinum genome showed that the genome does not contain complete insertion sequence (IS), only 6 and 5 of residual inserted sequence (partial IS). Using the codon preference analysis of genomic island (Genomic Island, GI), the results showed that two strains containing genomic island. These results showed that A (230611), F (230613) strain genome is relatively stable, and the life cycle of long-term in a state of dormancy. Bacillus virulence gene analysis showed that A (230611), F (230613) of 9 and 11 with bacterial adhesion and invasion related genes in the genome of Clostridium botulinum type and these genes contain adhesin (intimin), invasin (invasin), flagellar hook associated protein (Flagellar hook-associated) and other domains, and these genes also exist in other strains of the same type, may be related to the adhesion of Clostridium botulinum invasion of intestinal infection, The next step can be proved by experiments to prove the function of these genes.
And c.sporogenes genome alignment and the content of GC and recent source species does not produce botulinum toxin analysis shows that in the process of evolution, A (230611), F (230613) of Clostridium botulinum type in a specific location in the genome through lateral gene transfer, intake of a foreign gene fragment containing botulism clusters. In order to produce botulinum toxin of Clostridium sporogenes. At the same time compared with A1, A2, F (230613) of Clostridium botulinum toxin genome, we found three gene clusters of Clostridium botulinum were inserted into the chromosome of different loci. By genome comparison, we will insert the 9 loci of botulinum toxin gene cluster respectively. Type F (230613) of three loci. The genomic insertion site analysis showed that botulinum toxin gene cluster insert is not random, but regularly inserted into one of the 3 loci on chromosome.A1, B toxin gene cluster tend to plug In site 3, A2 tend to site 1, F (230613) tend to site 2. different types of toxin gene cluster structure shows that the non toxin gene with site-specific, and toxin gene mobility. We found that the bontF toxin gene cluster and site site 2 loci of bontA1 toxin gene recombination events the 3 cluster sites, the formation of bontA1 toxin gene cluster structure. The formation mechanism of the diversity of the botulinum toxin gene cluster.
The effect of the second part pH on the expression of A (230611) Clostridium botulinum toxin gene bontA and the regulation of the regulatory gene botR
Clostridium botulinum will experience acid in human intestinal infection in neutral and alkaline environment of pH. In order to understand the type of A (230611) of Clostridium botulinum toxin production of pH in different environments, we systematically measured pH (6.0,7.0,8.0,9.0) transcription of bacteria at different long time under the condition of bontA gene well, the secretion of toxin protein. In order to control the role of regulation of the toxin gene cluster in botR gene of bontA toxin gene, we also measured transcription of the botR gene, and the correlation between changes of bontA and botR mRNA. We further transcriptome sequencing of RNA culture in different growth periods of bacteria in different environmental conditions thus, in-depth study of toxin gene expression regulation mechanism.
At the transcriptional level, according to the bontA gene transcript levels indicated that transcription of pH 7 under the condition of the highest transcript levels in acidic and alkaline conditions significantly decreased (P0.01), transcription was the lowest under the condition of pH 9 (P0.01). It is suggested that bacteria in order to adapt to the acid and alkaline environment, can inhibit the expression of bontA gene at the transcriptional level at the protein level, the toxin protein secretion measured results showed that the toxin protein pH 7 under the condition of the secretion of the highest, pH 6 and pH 8 under the condition of reduced secretion, associated with toxin gene transcription is inhibited. But under the condition of pH9.0 toxin protein secretion and pH7.0 under the condition of no significant difference. The possible reason is: Although the pH value under the condition of 9 bontA gene transcription was inhibited, but after transcription translation or transfer efficiency increased.
Results show that the determination of botR gene transcription: transcription of botR gene in pH 7 under the condition of the highest transcript levels in acidic and alkaline conditions (P0.05), reduce the minimum quantity of transcription under the condition of pH 9 (P0.01) of.BotR gene and bontA gene expression correlation showed that the botR gene and bontA gene expression under different pH conditions is positive correlation in the growth process, the correlation coefficient between 0.7 and 1. This indicates that botR gene is the bontA gene, the growth cycle in bacteria, bontA gene transcription of botR gene transcription and the amount of collaborative change.
We selected a large quantity of toxin gene transcription of pH7 and pH9 two cultured bacteria, were obtained from the exponential phase, stationary phase and decline phase of bacteria were prepared by transcriptome sequencing, gene cluster analysis to find the co expression of transcription factors associated with toxin gene expression, cis acting elements, and the regulation of RNA, such as 5 'UTR, 3' UTR, small RNA and so on.

【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R378

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