【作者】 薛峰；

【导师】 严杰； X.Frank Yang； Jun Liu；

【作者基本信息】 浙江大学 ， 微生物学， 2011， 博士

【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。更多还原

【Abstract】 Leptospira interrogans is the major causative agent of leptospirosis, the most widespread zoonosis in the world. Recent studies show that phagocytosis plays an important role in the innate immune response to Leptospira infection, and L. interrogans can evade the killing of host phagocytes. However, little is known about the host adaptation of L. interrogans in this process.To better understand the molecular mechanisms during the initial interaction of pathogenic Leptospira and host innate immunity, we employed high-density microarray and comparative genomic approaches detecting transcriptomic changes in L. interrogans Serovar Lai Strain Lai 56601 using macrophage-derived cell models. Our results showed that upon interaction with host cells, L. interrogans dramatically altered expression of many genes involved in carbohydrate metabolism, energy production, lipid metabolism, signal transduction, transcription, translation, oxygen tolerance and outer membrane protein profile. Expression of the heme-containing catalase gene(katE) was significantly up-regulated, suggesting catalase may contribute to resisting the oxidative pressure of the macrophage. Furthermore, several major outer membrane protein (OMP) genes (e.g., ompLl, lipL32, lipL41, lipL48 and ompL47) were dramatically down-regulated (10-50 folds) upon interaction with macrophages, consistent with previous observations that OMPs are regulated in vivo. The persistent down-regulations of representative OMPs, including OmpLl, LipL32 and LipL41, were confirmed by immunoblotting. Lastly, to gain initial insight into the molecular mechanisms underlying differential gene expression in L. interrogans, we re-defined the putative transcriptional factors (TF) present in the genome and identified the major OmpR TF gene (LB333) is concurrently regulated with the down-regulated major OMP genes, suggesting a potential role of LB333 in regulation of these OMPs.This is the first report on transcriptional response of pathogenic Leptospira to host innate immunity and systemic analysis of TFs present in the Leptospira genomes. Alterations of the leptospiral OMP profile up interaction with host antigen-presenting cells (APC) provide critical information for selecting candidates as vaccine targets. Global surveys of the transcriptome primarily reveal the immune evasion strategies of L. interrogans in the early stage of infection and the potential TF vital for the reduction of the major OMPs. Leptospirosis is an important tropical disease around the world, particularly in humid tropical and subtropical countries. The pathogenic Leptospira can infect human through mucous membrane or abrasions in the skin, penetrate rapidly into blood stream, and diffuse into liver, lung, kidney and other organs. The clinical symptoms of different pathogenic Leptospira strains are complicated and complex, including hemorrhage, diarrhea, jaundice, severe renal impairment, and aseptic meningitis, etc. The high virulent strain, for example, Leptospira interrogans acutely infect human and cause severe organs failure. The mortality of the severe lung hemorrhage reach up to 15%.Recently, the innate immunity had been verified to be important for the host to response to the acute infection of leptospirosis. Pathogenic Leptospira can resist the phagocytosis and killing of macrophage, induce the apoptosis of macrophage. As one of the major immune cell of innate immunity, mature macrophage can phagocytize and kill pathogens, process and present antigens for adaptive immunity system, and secret a series of cytokines and chemokines to regulate host immune response. Leptospira interrogans was also found to survive and replicate in human macrophage, but was killed in murine macrophage, which may partially contribute the different severity of chronic infection in reservoir hosts and acute infection in human. To data, approaches to studying the capacity of pathogenic Leptospira to provoke macrophage response had been limited to individual genes and limited pathways. It is also expected to find more activated inflammatory genes and signaling pathways in macrophage infected by pathogenic Leptospira.In this study, we employed whole genome gene expression microarray and GO/KEGG gene function database analyzing the different responses of murine and human macrophages to pathogenic Leptospira interrogans serovar Lai strain Lai 56601. The comparative microarray results partially revealed the different innate immune responses between chronic infection in reservoir hosts and acute infection in humans. The gene regulations in antigen process and presentation of human cells are much less than those of murines. The key component of complement pathway, C3, was only significantly up-regulated in murine cells, which may contribute the complement activation process during the early stage of the infection in murine host. The CASP8 and FADD-like apoptosis regulator genes were significantly up-regulated, which strongly supported our previous results that the caspase-8/3 pathway plays an important role in apoptosis.It is very difficult to reveal the infection mechanism of leptospirosis, mainly due to the facts that the clinical symptoms of leptospirosis are complex, as well as the leptospiral components are complicated. This study revealed some transcriptional differences of the murine and human macrophage cell lines infected by L. interrogans, which set the foundation for further study on the innate immune response to leptospiral infection. The gene regulation system of Leptospira biflexa is much more complicated than that of Borrelia burgdoriferi, mainly due to the fact that L. biflexa has a large genome and more than 100 specific transcription factors (TF). There is no alternative Sigma S (RpoS) homolog in the genome of L. biflexa, which suggests that the Sigma N (Sigma54, RpoN) factor may play an important role in the gene regulation. However, the function of the RpoN of L. biflexa may be quite different from that of Borrelia burgdoriferi because L. biflexa has lots of specific TF.The RpoN gene of L. biflexa was inactivated by the homologous recombination gene knock method in this study. Then, the gene regulation profile of RpoN was analyzed by whole genome microarray. The RpoN of L. biflexa mainly regulated the genes of nitrogen metabolism, which is similar with RpoN function of the research model, Escherichia coli. The RpoN mutant utilized much less Tween-80 lipid component than the wild type. The Cryo-ET structural analysis and specific Nile red staining revealed that the RpoN can not synthesize the Poly-3-Hydroxybutyrate (PHB) storage, which may be associated with the microarray data that RpoN regulated the nitrogen utilize genes. In addition, the death rate of RpoN mutant in pure water is always higher than that of the wild type.This is the first molecular leptospiral research that an important alternative TF was inactivated by the target gene knock method. L. biflexa RpoN mutant lost the ability of synthesizing PHB storage and the high survivability in pure water, which indicated that leptospiral RpoN is essential for the resistant ability in low nutriment. Considering that the Sigma TF system of L. biflexa is almost similar with that of pathogenic L. interrogans, it is possible that the RpoN of L. interrogans is also essential for in vitro survivability and transmission. Leptospira interrogans is the primary causative agent of the most widespread zoonotic disease, leptospirosis. Despite its significance in global public health, the molecular biology and pathogenesis of L. interrogans are poorly understood, largely due to the lack of robust genetic techniques and in-depth structural insight of this pathogen. In this study, cryo-electron tomography was utilized to compare pathogenic and saprophytic species and uncover the unique biological and pathogenic features of Leptospira spp. A major difference between L. interrogans and L. biflexa is located at the lipopolysaccharide (LPS) layer, providing direct evidence that LPS varied considerably among leptospiral serovars. Novel periplasmic filaments might function as "skeleton" of the characteristic helical morphology. Bacterial DNA fingerprints captured in living organisms indicate that intracellular DNA forms tightly packed bundles (with～3.3nm spacing) in the middle of cylindrical body. A novel chemotaxis receptor array, a unique "hat" at cell end, as well as high resolution structure of in situ flagellar motor, provide not only the structural insights into the unique morphology of Leptospira spp. but also the plausible mechanism on attaching and penetrating mammalian cells during the spirochete-host interaction.更多还原