本文選題：問(wèn)號(hào)鉤體 + 巨噬細(xì)胞系��； 參考：《浙江大學(xué)》2011年博士論文

【摘要】：鉤端螺旋體病(Leptospirosis,鉤體病)世界范圍內(nèi)廣泛分布的人畜共患病。問(wèn)號(hào)鉤端螺旋體(Leptospira interrogans,問(wèn)號(hào)鉤體)是造成鉤體病的主要致病菌。最近研究表明,吞噬作用在宿主固有免疫系統(tǒng)抵抗鉤體感染中起到了重要作用,且問(wèn)號(hào)鉤體可以逃避吞噬細(xì)胞的殺傷作用。然而,該過(guò)程中問(wèn)號(hào)鉤體的宿主適應(yīng)性變化尚未可知。 為了研究致病性鉤體與宿主固有免疫互作過(guò)程中的分子機(jī)理,我們采用高密度基因芯片和比較基因組學(xué)方法研究了問(wèn)號(hào)鉤體賴型賴株56601在感染巨噬細(xì)胞系過(guò)程中的轉(zhuǎn)錄組學(xué)變化。結(jié)果表明,問(wèn)號(hào)鉤體在接觸細(xì)胞過(guò)程中迅速調(diào)控了許多生化途徑的基因表達(dá),涉及碳源代謝,能量產(chǎn)生,脂類代謝,信號(hào)傳導(dǎo),轉(zhuǎn)錄與翻譯,抗氧化,以及外膜蛋白等。含血紅素的過(guò)氧化氫酶基因(katE)顯著上調(diào)4-7倍,說(shuō)明該酶可能是對(duì)抗宿主氧化殺傷的的主要功能基因。另外,多個(gè)主要外膜蛋白的基因,如ompLl, lipL32, lipL41 lipL48, ompL47等,在接觸巨噬細(xì)胞的過(guò)程中顯著下調(diào)10-50倍,這與先前動(dòng)物模型中分離鉤體的外膜蛋白定量研究結(jié)果一致。本研究進(jìn)一步用免疫雜交方法驗(yàn)證了這些外膜蛋白在蛋白水平的持續(xù)下調(diào)。最后,結(jié)合比較基因組和基因組更新注釋,本研究重新定義分類了鉤體轉(zhuǎn)錄因子基因家族,并發(fā)現(xiàn)OmpR家族主要轉(zhuǎn)錄因子基因LB333的表達(dá)與主要外膜蛋白基因協(xié)同調(diào)控,初步說(shuō)明該因子可能參與主要外膜蛋白的調(diào)控。 本研究首次揭示了問(wèn)號(hào)鉤體應(yīng)對(duì)宿主固有免疫系統(tǒng)的全轉(zhuǎn)錄組變化,主要結(jié)果與先前體外條件調(diào)控轉(zhuǎn)錄組的研究結(jié)果有顯著差異。問(wèn)號(hào)鉤體在接觸宿主抗原遞呈細(xì)胞(APCs)過(guò)程中顯著改變了外膜系統(tǒng),這可能是重要的免疫逃逸機(jī)制,并為今后選擇亞單位疫苗靶點(diǎn)提供了重要參考信息。本研究重新定義了鉤體基因組中的轉(zhuǎn)錄因子基因,這為進(jìn)一步研究鉤體轉(zhuǎn)錄調(diào)控奠定了基礎(chǔ)。 鉤端螺旋體病(Leptospirosis,鉤體病)是世界范圍內(nèi)廣泛存在的熱帶病,尤其在濕熱的熱帶和亞熱帶地區(qū)較為流行。致病性鉤端螺旋體(Pathogenic Leptospira)通過(guò)粘膜或者傷口感染宿主后,迅速進(jìn)入血流并擴(kuò)散到肺,肝,腎和其他組織器官。不同致病性鉤體感染宿主的臨床癥狀復(fù)雜,包括眼結(jié)膜充血,腹瀉,黃疸,腎衰和腦膜炎等。強(qiáng)致病性鉤體,如問(wèn)號(hào)鉤體(Leptospira interrogans)等,所造成急性感染能導(dǎo)致嚴(yán)重的器官損傷。其中嚴(yán)重肺出血的致死率高達(dá)15%。 近幾年,固有免疫系統(tǒng)被證實(shí)在鉤體急性感染中起到了重要作用。致病性鉤體能夠逃避宿主巨噬細(xì)胞的吞噬,且誘導(dǎo)宿主細(xì)胞凋亡。巨噬細(xì)胞作為固有免疫系統(tǒng)中主要的免疫效應(yīng)細(xì)胞之一,在吞噬和殺滅,抗原遞呈以及免疫調(diào)節(jié)等方面起到了重要作用。問(wèn)號(hào)鉤體感染鼠類和人后,僅可以在人源巨噬細(xì)胞中存活并繁殖,這可能與問(wèn)號(hào)鉤體慢性感染鼠類和急性感染人類后不同的臨床癥狀呈正相關(guān)。目前研究致病性鉤體與宿主巨噬細(xì)胞互作的分子機(jī)制研究?jī)H停留在個(gè)別基因或者信號(hào)通路上,缺少全局性認(rèn)識(shí)。 本研究采用本論文第一部分的鉤體感染巨噬細(xì)胞系模型和全轉(zhuǎn)錄組基因芯片技術(shù),結(jié)合GO和KEGG基因功能和分類數(shù)據(jù)庫(kù),初步揭示了問(wèn)號(hào)鉤體賴型賴株56601感染鼠源和人源巨噬細(xì)胞系后基因轉(zhuǎn)錄水平的整體差異。通過(guò)比較轉(zhuǎn)錄組學(xué)研究,發(fā)現(xiàn)兩類細(xì)胞在炎癥因子和趨化因子表達(dá)上存在較大差異；人源巨噬細(xì)胞系的抗原遞呈途徑基因受調(diào)控幅度明顯小于鼠源細(xì)胞；發(fā)現(xiàn)補(bǔ)體途徑中處于核心地位的C3組分在鼠源細(xì)胞中顯著上調(diào),這可能有助于鼠類宿主感染初期的補(bǔ)體激活；凋亡途徑中,CASP8凋亡調(diào)控基因的顯著上調(diào),這與本實(shí)驗(yàn)室已發(fā)表的問(wèn)號(hào)鉤體通過(guò)caspase-8釉caspase-3通路誘導(dǎo)宿主細(xì)胞凋亡的結(jié)論一致。 鉤體病臨床癥狀多樣,致病型鉤體菌體物質(zhì)成分復(fù)雜,這都決定了鉤體感染研究是一項(xiàng)任重道遠(yuǎn)的工作。本研究通過(guò)高通量基因表達(dá)譜篩選和生物途徑統(tǒng)計(jì)分析,發(fā)現(xiàn)了一些鉤體感染鼠源和人源巨噬細(xì)胞系后表達(dá)水平的差異,為進(jìn)一步深入研究鉤體感染的固有免疫機(jī)理奠定了基礎(chǔ)。 雙曲鉤端螺旋體(Leptospira biflexa)的轉(zhuǎn)錄調(diào)控系統(tǒng)較伯氏疏螺旋體(Borrelia burgdoriferi)更為復(fù)雜,主要表現(xiàn)為基因組較大,編碼100多個(gè)特異性轉(zhuǎn)錄因子(specific TF).雙曲鉤體沒(méi)有非特異性Sigma S (RpoS)轉(zhuǎn)錄因子,所以Sigma N (Sigma54, RpoN)轉(zhuǎn)錄因子可能在轉(zhuǎn)錄調(diào)控中起到了更大的作用。但由于鉤體特異性轉(zhuǎn)錄因子較多,鉤體RpoN在轉(zhuǎn)錄調(diào)控中的作用可能與伯氏疏螺旋體的RpoN大不相同。 本研究采用同源重組基因敲除的方法,定點(diǎn)失活了雙曲鉤體的RpoN轉(zhuǎn)錄因子。進(jìn)一步采用全轉(zhuǎn)錄組基因芯片技術(shù)檢測(cè)了該轉(zhuǎn)錄因子的調(diào)控靶點(diǎn)。雙曲鉤體RpoN的調(diào)控靶點(diǎn)主要是氮源代謝相關(guān)基因,與模式生物大腸桿菌相似。野生株和突變體的培養(yǎng)物中吐溫-80成分的消耗量差異顯著,突變體的消耗量大幅度減少。通過(guò)Cryo-ET分子電鏡結(jié)構(gòu)分析和尼羅紅染色,發(fā)現(xiàn)雙曲鉤體RpoN突變體已經(jīng)不再形成聚beta-羥基丁酸(PHB)貯藏物。這可能與RpoN正調(diào)控氨攝取基因有關(guān)。另外,RpoN突變體在純水中的死亡速度明顯快于野生株。 本研究是鉤端螺旋體上第一項(xiàng)定點(diǎn)敲除轉(zhuǎn)錄因子研究其調(diào)控機(jī)理的分子細(xì)菌學(xué)研究。雙曲鉤體RpoN突變體喪失了合成貯藏物的能力,并且在純水中的生存能力也明顯下降。這說(shuō)明RpoN轉(zhuǎn)錄因子對(duì)于雙曲鉤體在無(wú)養(yǎng)料條件下的耐受能力至關(guān)重要的�？紤]到問(wèn)號(hào)鉤體與雙曲鉤體的Sigma非特異性轉(zhuǎn)錄調(diào)控系統(tǒng)基本相同,可以推測(cè)問(wèn)號(hào)鉤體的RpoN也是正調(diào)控其體外生長(zhǎng)傳播能力的主要因子。 鉤體病(Leptospirosis)是世界范圍內(nèi)廣泛分布的人畜共患病。問(wèn)號(hào)鉤體(Leptospira interrogans)是造成該傳染病的主要致病菌。目前,致病性鉤體仍然缺乏有效的基因操作手段,且其結(jié)構(gòu)生物學(xué)研究尚未開展,所以該致病菌的分子生物學(xué)特性和該全球性公共疾病的致病機(jī)理尚不明了。本研究采用低溫電鏡技術(shù)(Cryo-electron tomography, Cryo-ET)比較分析致病性問(wèn)號(hào)鉤體和腐生性雙曲鉤體的精細(xì)結(jié)構(gòu),揭示了鉤體的一些生物學(xué)新特性和可能的致病機(jī)理。問(wèn)號(hào)與雙曲鉤體的主要區(qū)別是兩者有不同含量的LPS成分。這初步證明了鉤體LPS含量在不同血清型之間差別很大的推論。鉤體獨(dú)特的胞質(zhì)纖維成分可能是決定鉤體特有的螺旋狀形態(tài)的骨架。冰凍活鉤體的DNA成分在柱狀細(xì)胞內(nèi)呈緊密束狀存在,折疊的間隙約為3.3nm。另外,本研究也揭示了雙曲鉤體特有的甲基接收蛋白結(jié)構(gòu),鉤體特有的頂端“帽子”結(jié)構(gòu),和鉤體鞭毛馬達(dá)的高精細(xì)結(jié)構(gòu)。這些新發(fā)現(xiàn)不僅闡明了鉤體的獨(dú)特的結(jié)構(gòu)和形態(tài),也為研究鉤體與宿主互作過(guò)程中的感染機(jī)制奠定了基礎(chǔ)。
[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.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2011
【分類號(hào)】：R377

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相關(guān)期刊論文 前1條

1 ;Identification and classification of all potential hemolysin encoding genes and their products from Leptospira interrogans serogroup Icterohaemorrhagiae serovar Lai[J];Acta Pharmacologica Sinica;2005年04期

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本文編號(hào)：1903910