钩端螺旋体感染的转录组学与钩体结构生物学研究

发布时间：2018-05-18 02:12

本文选题：问号钩体 + 巨噬细胞系　；参考：《浙江大学》2011年博士论文

【摘要】：钩端螺旋体病(Leptospirosis,钩体病)世界范围内广泛分布的人畜共患病。问号钩端螺旋体(Leptospira interrogans,问号钩体)是造成钩体病的主要致病菌。最近研究表明,吞噬作用在宿主固有免疫系统抵抗钩体感染中起到了重要作用,且问号钩体可以逃避吞噬细胞的杀伤作用。然而,该过程中问号钩体的宿主适应性变化尚未可知。为了研究致病性钩体与宿主固有免疫互作过程中的分子机理,我们采用高密度基因芯片和比较基因组学方法研究了问号钩体赖型赖株56601在感染巨噬细胞系过程中的转录组学变化。结果表明,问号钩体在接触细胞过程中迅速调控了许多生化途径的基因表达,涉及碳源代谢,能量产生,脂类代谢,信号传导,转录与翻译,抗氧化,以及外膜蛋白等。含血红素的过氧化氢酶基因(katE)显著上调4-7倍,说明该酶可能是对抗宿主氧化杀伤的的主要功能基因。另外,多个主要外膜蛋白的基因,如ompLl, lipL32, lipL41 lipL48, ompL47等,在接触巨噬细胞的过程中显著下调10-50倍,这与先前动物模型中分离钩体的外膜蛋白定量研究结果一致。本研究进一步用免疫杂交方法验证了这些外膜蛋白在蛋白水平的持续下调。最后,结合比较基因组和基因组更新注释,本研究重新定义分类了钩体转录因子基因家族,并发现OmpR家族主要转录因子基因LB333的表达与主要外膜蛋白基因协同调控,初步说明该因子可能参与主要外膜蛋白的调控。本研究首次揭示了问号钩体应对宿主固有免疫系统的全转录组变化,主要结果与先前体外条件调控转录组的研究结果有显著差异。问号钩体在接触宿主抗原递呈细胞(APCs)过程中显著改变了外膜系统,这可能是重要的免疫逃逸机制,并为今后选择亚单位疫苗靶点提供了重要参考信息。本研究重新定义了钩体基因组中的转录因子基因,这为进一步研究钩体转录调控奠定了基础。钩端螺旋体病(Leptospirosis,钩体病)是世界范围内广泛存在的热带病,尤其在湿热的热带和亚热带地区较为流行。致病性钩端螺旋体(Pathogenic Leptospira)通过粘膜或者伤口感染宿主后,迅速进入血流并扩散到肺,肝,肾和其他组织器官。不同致病性钩体感染宿主的临床症状复杂,包括眼结膜充血,腹泻,黄疸,肾衰和脑膜炎等。强致病性钩体,如问号钩体(Leptospira interrogans)等,所造成急性感染能导致严重的器官损伤。其中严重肺出血的致死率高达15%。近几年,固有免疫系统被证实在钩体急性感染中起到了重要作用。致病性钩体能够逃避宿主巨噬细胞的吞噬,且诱导宿主细胞凋亡。巨噬细胞作为固有免疫系统中主要的免疫效应细胞之一,在吞噬和杀灭,抗原递呈以及免疫调节等方面起到了重要作用。问号钩体感染鼠类和人后,仅可以在人源巨噬细胞中存活并繁殖,这可能与问号钩体慢性感染鼠类和急性感染人类后不同的临床症状呈正相关。目前研究致病性钩体与宿主巨噬细胞互作的分子机制研究仅停留在个别基因或者信号通路上,缺少全局性认识。本研究采用本论文第一部分的钩体感染巨噬细胞系模型和全转录组基因芯片技术,结合GO和KEGG基因功能和分类数据库,初步揭示了问号钩体赖型赖株56601感染鼠源和人源巨噬细胞系后基因转录水平的整体差异。通过比较转录组学研究,发现两类细胞在炎症因子和趋化因子表达上存在较大差异；人源巨噬细胞系的抗原递呈途径基因受调控幅度明显小于鼠源细胞；发现补体途径中处于核心地位的C3组分在鼠源细胞中显著上调,这可能有助于鼠类宿主感染初期的补体激活；凋亡途径中,CASP8凋亡调控基因的显著上调,这与本实验室已发表的问号钩体通过caspase-8釉caspase-3通路诱导宿主细胞凋亡的结论一致。钩体病临床症状多样,致病型钩体菌体物质成分复杂,这都决定了钩体感染研究是一项任重道远的工作。本研究通过高通量基因表达谱筛选和生物途径统计分析,发现了一些钩体感染鼠源和人源巨噬细胞系后表达水平的差异,为进一步深入研究钩体感染的固有免疫机理奠定了基础。双曲钩端螺旋体(Leptospira biflexa)的转录调控系统较伯氏疏螺旋体(Borrelia burgdoriferi)更为复杂,主要表现为基因组较大,编码100多个特异性转录因子(specific TF).双曲钩体没有非特异性Sigma S (RpoS)转录因子,所以Sigma N (Sigma54, RpoN)转录因子可能在转录调控中起到了更大的作用。但由于钩体特异性转录因子较多,钩体RpoN在转录调控中的作用可能与伯氏疏螺旋体的RpoN大不相同。本研究采用同源重组基因敲除的方法,定点失活了双曲钩体的RpoN转录因子。进一步采用全转录组基因芯片技术检测了该转录因子的调控靶点。双曲钩体RpoN的调控靶点主要是氮源代谢相关基因,与模式生物大肠杆菌相似。野生株和突变体的培养物中吐温-80成分的消耗量差异显著,突变体的消耗量大幅度减少。通过Cryo-ET分子电镜结构分析和尼罗红染色,发现双曲钩体RpoN突变体已经不再形成聚beta-羟基丁酸(PHB)贮藏物。这可能与RpoN正调控氨摄取基因有关。另外,RpoN突变体在纯水中的死亡速度明显快于野生株。本研究是钩端螺旋体上第一项定点敲除转录因子研究其调控机理的分子细菌学研究。双曲钩体RpoN突变体丧失了合成贮藏物的能力,并且在纯水中的生存能力也明显下降。这说明RpoN转录因子对于双曲钩体在无养料条件下的耐受能力至关重要的。考虑到问号钩体与双曲钩体的Sigma非特异性转录调控系统基本相同,可以推测问号钩体的RpoN也是正调控其体外生长传播能力的主要因子。钩体病(Leptospirosis)是世界范围内广泛分布的人畜共患病。问号钩体(Leptospira interrogans)是造成该传染病的主要致病菌。目前,致病性钩体仍然缺乏有效的基因操作手段,且其结构生物学研究尚未开展,所以该致病菌的分子生物学特性和该全球性公共疾病的致病机理尚不明了。本研究采用低温电镜技术(Cryo-electron tomography, Cryo-ET)比较分析致病性问号钩体和腐生性双曲钩体的精细结构,揭示了钩体的一些生物学新特性和可能的致病机理。问号与双曲钩体的主要区别是两者有不同含量的LPS成分。这初步证明了钩体LPS含量在不同血清型之间差别很大的推论。钩体独特的胞质纤维成分可能是决定钩体特有的螺旋状形态的骨架。冰冻活钩体的DNA成分在柱状细胞内呈紧密束状存在,折叠的间隙约为3.3nm。另外,本研究也揭示了双曲钩体特有的甲基接收蛋白结构,钩体特有的顶端“帽子”结构,和钩体鞭毛马达的高精细结构。这些新发现不仅阐明了钩体的独特的结构和形态,也为研究钩体与宿主互作过程中的感染机制奠定了基础。
[Abstract]:Leptospirosis (Leptospirosis, Leptospira) is widely distributed worldwide in zoonosis. The Leptospira interrogans (Leptospira interrolea) is the main pathogenic bacteria causing leptospirosis. Recent studies have shown that phagocytosis plays an important role in the host innate immune system resistance to leptospirosis, and the interrogation hook The body can evade the killing effect of phagocytic cells. However, the host adaptive change of the interrogation of Leptospira interna is unknown.
In order to study the molecular mechanism of the inherent immune interaction between the pathogenic Leptospira and the host, we studied the transcriptional changes of the Leptospira Lai 56601 in the process of macrophage infection by high density gene chip and comparative genomics. The results showed that the Leptospira was rapidly regulated in the process of contact cells. The gene expression of multiple biochemical pathways involves carbon source metabolism, energy production, lipid metabolism, signal transduction, transcription and translation, antioxidant, and outer membrane proteins. The heme containing catalase gene (katE) is up to 4-7 times up, indicating that the enzyme may be the main functional gene against the oxygenation of the host. In addition, several major outer membrane proteins The genes, such as ompLl, lipL32, lipL41 lipL48, ompL47, and so on, were significantly down regulated by 10-50 times in contact with macrophages, which were consistent with the results of the quantitative study of the outer membrane proteins of the isolated Leptospira in previous animal models. Compared with genomic and genomic update annotations, this study redefined the gene family of Leptospira transcriptional factor (Leptospira), and found that the expression of the main transcription factor gene LB333 in the OmpR family was coordinated with the main outer membrane protein genes. It was preliminarily indicated that the factor may be involved in the regulation of the main outer membrane proteins.
This study was the first to reveal the changes in the whole transcriptional group of the Leptospira's response to the host immune system for the first time. The main results were significantly different from that of the previous studies. The Leptospira significantly changed the outer membrane system during contact with the host antigen presenting cell (APCs), which may be an important immune escape mechanism. This study redefines the transcription factor genes in the Leptospira genome, which provides a basis for further study of Leptospira transcriptional regulation.
Leptospirosis (Leptospirosis) is a widespread tropical disease worldwide, especially in hot and hot tropical and subtropical regions. The pathogenic Leptospira (Pathogenic Leptospira) rapidly enters the blood flow and spreads to the lungs, liver, kidney and other tissues and organs after infection of the host by mucous membrane or wound. The clinical symptoms of the host of pathogenic Leptospira are complex, including conjunctival congestion, diarrhea, jaundice, renal failure and meningitis. Strong pathogenic Leptospira (Leptospira interrogans), such as the Leptospira (the interroptospira), can cause severe organ damage. The fatal rate of severe pulmonary blood is up to 15%.
In recent years, the inherent immune system has been proved to play an important role in the acute infection of the leptospira. The pathogenic Leptospira can escape the phagocytosis of the host macrophage and induce the apoptosis of the host cells. As one of the main immune effector cells in the inherent immune system, macrophages are phagocytic and killed, antigen presentation and immunoregulation. The Leptospira of the Leptospira can only survive and reproduce in human macrophages, which may be positively related to the different clinical symptoms of the chronic infection of the rodent with the leptospira and the acute infection of the human being. The study of the molecular mechanism of the interaction of the pathogenic Leptospira with the host macrophage cell is only in a few cases. There is a lack of global awareness on genes or signaling pathways.
In this study, using the first part of this study, the macrophage model of leptospiral infection and the whole transcriptional gene chip technology, combined with the function of GO and KEGG gene and the classification database, the whole difference of gene transcription of the gene transcription of the Leptospira Lai 56601 infected rat and human macrophage system was preliminarily revealed by comparative transcriptional study. It was found that there were significant differences in the expression of inflammatory factors and chemokines in the two types of cells. The antigen presenting pathway of the human macrophage system was significantly less regulated than that of the mouse, and it was found that the C3 component at the core in the complement pathway was significantly up-regulated in the mouse source cells, which may help to supplement the early infection of the rat host infection. Body activation; in the apoptotic pathway, the apoptosis regulation gene of CASP8 is significantly up-regulated, which is consistent with the conclusion that the Leptospira has been induced by the caspase-8 glaze caspase-3 pathway published in our laboratory.
The clinical symptoms of Leptospira are diverse and the substance components of the pathogenic Leptospira are complex, which determines that the study of Leptospira infection is a task with a long way to go. This study found the difference in the expression level of some leptospiral infection rats and human macrophages by high throughput gene expression spectrum screening and biological pathway analysis. It lays a foundation for further study of the innate immune mechanism of leptospirosis.
The transcriptional regulation system of Leptospira biflexa is more complex than Borrelia burgdoriferi, which is mainly characterized by large genome, encoding more than 100 specific transcription factors (specific TF). The hyperbolic Leptospira has no non specific Sigma S (RpoS) transcription factors, so Sigma N (Sigma54,) transcription factors It may play a greater role in transcriptional regulation, but the role of leptospiral RpoN in transcriptional regulation may be different from the RpoN of Borrelia burgdorferi due to the large number of leptospiral specific transcription factors.
In this study, the homologous recombination gene knockout method was used to inactivate the RpoN transcription factor of the hyperbolic Leptospira. Further, the transcriptional gene chip technology was used to detect the regulatory target of the transcription factor. The target of the control target of the RpoN of the hyperbolic Leptospira was mainly the nitrogen source related genes, similar to the model biological Escherichia coli, and the wild strain and mutation. The consumption of Twain -80 components was significantly different in the body culture, and the consumption of the mutant was greatly reduced. The Cryo-ET molecular electron microscope structure analysis and Nile red staining showed that the hyperbolic mutant of the Leptospira RpoN had no longer Formed Poly beta- hydroxybutyric acid (PHB) storage. This may be related to RpoN positive regulation of the ammonia uptake gene. In addition, RpoN mutation The death rate of the body in pure water is faster than that in wild plants.
This study is a molecular bacteriological study on the regulatory mechanism of the first fixed-point knockout factor on the leptospira. The RpoN mutant of the hyperbolic Leptospira lost the ability to synthesize storage and decreased the viability in pure water. This shows that the RpoN transcription factor has the tolerance to the double Leptospira under the condition of no feed. It is important that the Sigma nonspecific transcriptional regulation system of the leptospira and the hyperbolic Leptospira are basically the same, and it is possible to speculate that the RpoN of the Leptospira is also the main factor that regulates the growth and propagation ability of the leptospira.
Leptospirosis (Leptospirosis) is widely distributed worldwide and zoonosis. The Leptospira interrogans is the main pathogenic bacteria causing the infectious disease. At present, the pathogenic Leptospira is still lack of effective gene manipulation methods, and its structural biology has not been carried out, so the molecular biological characteristics of the pathogenic bacteria and the biological characteristics of the pathogenic bacteria The pathogenesis of global public diseases is still unknown. This study compared the fine structures of the pathogenic Leptospira and the rotten hyperbolic Leptospira by Cryo-electron tomography (Cryo-ET), and revealed some new biological characteristics and possible pathogenesis of the leptospira. The main difference between the question mark and the hyperbolic Leptospira is two There are different content of LPS components. This preliminarily proves that the LPS content of the Leptospira is very different between different serotypes. The unique cytoplasmic fiber components of the Leptospira may be the skeleton of the spiral shape peculiar to the leptospira. The DNA components of the frozen Leptospira are closely bundled in the columnar cells, and the folding space is about 3.3nm.. This study also revealed the structure of the hyperbolic leptospirosis, the top "hat" structure of the Leptospira, and the high fine structure of the Leptospira flagellum. These new discoveries not only elucidate the unique structure and morphology of the Leptospira, but also lay the foundation for the study of the mechanism of infection in the intercourse of the leptospira and the host.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R377

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