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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 林俊宏(Chun-Hung Lin) | |
dc.contributor.author | Hau-Ming Jan | en |
dc.contributor.author | 詹皓名 | zh_TW |
dc.date.accessioned | 2021-06-08T01:46:49Z | - |
dc.date.copyright | 2016-08-24 | |
dc.date.issued | 2016 | |
dc.date.submitted | 2016-08-09 | |
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Niemann, Journal of molecular biology, 2015, 427, 1304-1315. | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/19152 | - |
dc.description.abstract | 世界上大約有一半的人口被胃幽門螺旋桿菌所感染,而這個細菌也是造成各種胃部疾病的主要原因。這種病菌是無法製造膽固醇的,它會吸收宿主細胞身上的膽固醇並將其轉換成各種膽固醇醣苷衍生物,包括膽固醇6'-酰基和6'-磷脂基葡萄糖苷(CAG 和CPG)。由於缺乏靈敏的分析方法,無法得知在CAG 和CPG 上面的酰基(acyl)或是磷脂基(phosphatidyl)的組成和變化,是否會影響在生理上所扮演的角色。在此,我們建立了一個達到飛莫爾(femto-molar)層級的代謝物標記法,並利用這樣的方式將這些衍生物做詳細的定性和定量的分析,得以建立膽固醇醣苷衍生物的質譜/質譜資料庫,並可分析所有的膽固醇代謝衍生物。進一步發現這些細菌能夠獲得宿主表皮細胞的磷脂,進行CAG 的合成。同時,我們也找到負責CAG 生合成的酵素(HP0499),進一步證實HP0499 在感染宿主細胞時,會透過外膜小囊(OMV)或直接接觸的方式,將酵素及產物轉置到人類細胞上,以產生帶有較長或是較多不飽和酰基的CAG(又稱作含有人類脂質的CAG),因而促進脂筏(lipid rafts)的形成,以及細胞表面的整合素α5β1 的聚集,導致增加細菌黏附到宿主表皮細胞,因而大幅增加毒素蛋白因子(CagA)轉置進入宿主細胞。這樣的結果支持了一個想法: 幽門螺桿菌演化出特殊的宿主/病原體的交互作用以增加細菌的毒性。我們的研究也詳細闡述了,不同酰基組成的CAG,如何連結到細菌的致病性。 | zh_TW |
dc.description.abstract | Helicobacter pylori, which infects approximately half of the human population, is the main cause of various gastric diseases. This pathogen is auxotrophic for cholesterol, which it converts upon uptake to various cholesteryl alpha-glucoside derivatives, including cholesteryl 6’-acyl and 6’-phosphatidyl alpha-glucosides (CAGs and CPGs). Due to a lack of sensitive analytical methods, it remains unknown whether CAGs and CPGs have distinct physiological roles and how the acyl chain components affect function. Herein we describe a metabolite-labeling method for qualitatively and quantitatively characterizing these derivatives at a femto-molar detection limit. We generated an MS/MS database of CGds that allows for profiling of all cholesterol-derived metabolites. The subsequent analysis led to the unprecedented discovery that these bacteria acquire phospholipids for CAG biosynthesis from the membrane of epithelial cells. Furthermore, we also identified the enzyme responsible for CAG biosynthesis (HP0499). The result suggested that HP0499 and the resulting products are translocated from the bacterium to the host cell via direct contact or delivery by outer membrane vesicles (OMVs) to facilitate formation of longer and/or unsaturated CAG acyl chains (also called human lipid-containing CAGs), which helped to promote lipid raft clustering, induced accumulation of integrin α5β1, and thus enhanced bacterial adhesion. This also explained the enhanced translocation of the virulence factor CagA into the host cell. These findings demonstrate how H. pylori evolves the special host/pathogen interplay to enhance bacterial virulence, and that our research pinpoints an important connection between the CAG composition and bacterial pathogenicity. | en |
dc.description.provenance | Made available in DSpace on 2021-06-08T01:46:49Z (GMT). No. of bitstreams: 1 ntu-105-D99b46004-1.pdf: 14379791 bytes, checksum: d81301ccbb0ee65d861da8afcc9b0fab (MD5) Previous issue date: 2016 | en |
dc.description.tableofcontents | 口試委員會審定書 #
致謝 i 中文摘要 iii ABSTRACT iv CONTENTS vi LIST OF FIGURES x LIST OF TABLES xiv 縮寫表 xv Chapter 1 Introduction 1 1.1 Role of Helicobacter pylori in the pathogensis 1 1.2 Cholesterol glucosides 3 1.3 The biological function of cholesterol glucosides in the immune response 4 1.4 The biological function of cholesterol glucosides in pathogenesis 6 1.5 Goals of this thesis 12 Chapter 2 Results 14 2.1 Synthesis and evaluation of tagged metabolites 14 2.1.1 Synthesis of cholesterol analogues 14 2.1.2 Evaluation of tagged metabolites in vitro 14 2.1.3 Evaluation of tagged metabolites in vivo 15 2.1.4 Synthesis of alkyne-containing fluorophores 15 2.1.5 Examination of the yield and stability of alkyne containing fluorophore 15 2.2 Structural determination and quantification of CGds 16 2.2.1 Structural determination of MAN-labeled CGds by LC-MS and tandom MS analysis 16 2.2.2 Structural determination of MAN-labeled CGds by NMR analysis 17 2.2.3 Structural determination of MAN-labeled CGds by enzymatic analysis and 31P NMR spectrometry 17 2.2.4 Quantification of MAN-labeled CGds by HPLC 18 2.3 Uptake of human phospholipids for CAG biosynthesis in H. pylori 19 2.3.1 Composition analysis of CAGs in bacteria culture and co-culture 19 2.3.2 Composition analysis of CAGs in standard type H. pylori and clinical strains from patients with gastric cancer. 19 2.3.3 Utilization of human lipids for CAG and CPG biosynthesis in H. pylori 20 2.4 Identification of CAG synthase and determination of its location 21 2.4.1 Identification of CAG synthase 21 2.4.2 Localization of HP0499 for synthesis CAG 22 2.5 Investigate the effect of each derivative of CGds on pathogenesis 24 2.6 Characterization of the role of CAGs in bacterial virulence 24 2.6.1 Influence of CAGs on lipid raft clustering 25 2.6.2 Influence of CAG on CagA translocation 26 2.6.3 Influence of CAGs on bacterial adhesion 27 Chapter 3 Discussion and conclusion 30 3.1 Development of metabolic labeling to profile cholesterol glucoside derivatives 30 3.2 H. pylori acquires phospholipids from the host for CAG biosynthesis. 30 3.3 HP0499 is transferred to the surface of host cell 31 3.4 Among CGds, CAG play a major function in pathogenesis 33 3.5 Incorporation of human lipid-containing CAG could enhance lipid rafts clustering 34 3.6 Integrin in lipid rafts could enhance bacterial adhesion and CagA translocation 35 3.7 Conclusion 37 Chapter 4 Methods and materials 38 4.1 General consideration of chemical synthesis 38 4.2 Chemical synthesis 40 4.3 NMR spectra data 54 4.4 H. pylori strains and bacterial culture 57 4.5 Coupling assay for cholesterol glucosyltransferase activity 57 4.6 Quantitation of phospholipids 57 4.7 Metabolic labeling of CGds 58 4.8 HPLC analysis 58 4.9 UPLC-MS analysis 59 4.10 Phospholipase hydrolysis 60 4.11 31P NMR spectroscopic measurements 61 4.12 Co-culture experiments 61 4.13 Immunoprecipitation and blotting 63 4.14 Live-cell imaging of AGS cells 64 4.15 Immunofluorescence microscopy 64 4.16 Flow cytometry 65 References 67 Figures, Table and Legends 71 Publications 131 | |
dc.language.iso | en | |
dc.title | 膽固醇醣苷代謝物標定與分析以揭露它們如何增強胃幽門螺旋桿菌的致病性 | zh_TW |
dc.title | Metabolic labeling and profiling of cholesteryl glucosides to unveil how they enhance virulence in Helicobacter pylori | en |
dc.type | Thesis | |
dc.date.schoolyear | 104-2 | |
dc.description.degree | 博士 | |
dc.contributor.oralexamcommittee | 吳明賢(Ming-shiang wu),王雯靜(Wen-Ching Wang),陳逸然(Ya-Jen Chang),張雅貞(Ya-Jen Chang),高茂傑(Mou-Chieh Kao) | |
dc.subject.keyword | 胃幽門螺旋桿菌,膽固醇,代謝物,質譜分析,脂筏,磷脂, | zh_TW |
dc.subject.keyword | Helicobacter pylori,cholesterol,metabolite,mass analysis,lipid rafts,phospholipid, | en |
dc.relation.page | 143 | |
dc.identifier.doi | 10.6342/NTU201602064 | |
dc.rights.note | 未授權 | |
dc.date.accepted | 2016-08-09 | |
dc.contributor.author-college | 生命科學院 | zh_TW |
dc.contributor.author-dept | 生化科學研究所 | zh_TW |
顯示於系所單位: | 生化科學研究所 |
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