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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 | Yi-Chi Chen | en |
dc.contributor.author | 陳奕齊 | zh_TW |
dc.date.accessioned | 2021-06-16T09:47:47Z | - |
dc.date.available | 2022-02-16 | |
dc.date.copyright | 2017-02-16 | |
dc.date.issued | 2017 | |
dc.date.submitted | 2017-01-22 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/59966 | - |
dc.description.abstract | 胃幽門螺旋桿菌會引發許多胃部疾病,嚴重者會導致胃癌。在近年來的報導中發現,胃幽門螺旋桿菌所產生的膽固醇葡萄糖衍生物會促使胃幽門螺旋桿菌逃離宿主免疫反應。在剔除膽固醇葡萄糖衍生物的菌株中,其附著及經由第四型分泌系統造成的細胞毒素相關基因A轉移能力都大幅下降。因此,膽固醇葡萄糖衍生物被預期可增強胃幽門螺旋桿菌的附著及感染能力。
但在現階段下,要定性定量膽固醇葡萄糖衍生物是非常不容易的,且靈敏度也很差。因此,建立一個高靈敏度且快速方便的方法便是第一要務。在此研究中,我們合成了疊氮基膽固醇類似物,並使用於胃幽門螺旋桿菌培養中。在萃取及點擊反應將炔基螢光團修飾於疊氮基膽固醇葡萄糖衍生類似物上後,此化合物在質譜儀中的離子化能力便會大幅增強。其帶有的螢光性質也可以經由高效能液相層析儀分析並做純化,以利後續實驗使用。 在此基礎上,酵素的鑑定和活性功能的探討便可做深入的研究。因為在參與膽固醇葡萄糖衍生物的生合成酵素中,只有第一步的膽固醇葡萄糖基轉移酶被發現。剔除此基因會造成膽固醇葡萄糖衍生物完全缺乏,因此無法鑑定是哪一個衍生物會造成影響,所以我們對於下游疑似膽固醇葡萄糖醯基轉移酶及膽固醇葡萄糖磷脂基轉移酶都做了基因剔除的實驗。在同時利用基因剔除實驗和我們建立的分析方式之下,我們發現了膽固醇葡萄糖醯基轉移酶,而膽固醇葡萄糖磷脂基轉移酶的鑑定仍在進行中。在剔除此基因的菌株中,膽固醇醯基葡萄糖將不再被產生。此外,經由表現此重組蛋白,也可以在試管中產生膽固醇醯基葡萄糖,再次確認了此酵素的功能。此酵素先前被報導為一磷脂水解酶,在此論文中,我們不僅確認了其第二種功能,即膽固醇葡萄糖醯基轉移能力,也對其特性做了初步的探討,例如合適酸鹼值、受質專一性、酵素動力學探討等等,也將反應條件調整使之活性最大化。 實驗結果顯示,此酵素在中性至酸性環境下皆可有不錯的活性,其受質則偏好利用磷脂醯乙醇胺,而醯基之鏈長則沒有特別喜好。此現象暗示此酵素不只是在胃幽門螺旋桿菌上、在胃腔中、及在胃上皮細胞上均可有高度的活性。在探討此酵素存在區域的實驗中發現,其不僅是存在於胃幽門螺旋桿菌之外膜表面,也會被以外膜囊泡的方式釋放至菌體外,進而使其傳遞至細胞上。我們利用加入重組酵素及外膜囊泡的方式,偵測出在此酵素傳遞至細胞上後,會產生膽固醇醯基葡萄糖,並且造成細胞的脂質筏聚集,進而使胃幽門螺旋桿菌的附著力增加及細胞毒素相關基因A轉移增加。另外,在加入阻斷整合素抗體的實驗中,儘管膽固醇醯基葡萄糖有使脂質筏聚集,但胃幽門螺旋桿菌的附著力及細胞毒素相關基因A轉移皆被抑制,代表這些聚集的脂質筏是藉由吸引整合素,而使得胃幽門螺旋桿菌的附著力增加。 另外,此膽固醇葡萄糖醯基轉移酶的抑制劑也被發現。在存在此抑制劑的條件下,胃幽門螺旋桿菌的附著力及細胞毒素相關基因A轉移也皆被抑制,代表此抑制劑有治療胃幽門螺旋桿菌的潛力。藉由同時提供抗生素及膽固醇葡萄糖醯基轉移酶抑制劑,即可能可以處理棘手的多重抗藥性胃幽門螺旋桿菌。此研究提供了我們許多資訊以利後續之研究,同時也提供了一種可能的治療方式根除胃幽門螺旋桿菌,進而降低罹患胃癌的風險,保障胃部的健康。 | zh_TW |
dc.description.abstract | Helicobacter pylori infection represents the major cause in gastric pathology, such as ulcer and cancer. It is known that the bacteria cannot produce cholesterol and have to hijack cholesterol from the environment or the host cells. Upon uptake of cholesterol, H. pylori can produce cholesterol glucoside derivatives (CGds) biosynthetically that are known to impede phagocytosis and T cell activation. In addition, CGd deficiency was found to reduce type IV secretion system (T4SS)-associated activities and CagA translocation, suggesting that CGds influence host cell membrane and participate in H. pylori adhesion.
However, the involved enzymes and functional roles of these cholesterol-derived metabolites remain ambiguous, mainly owing to their low abundance and heterogeneous features. Herein, we developed a profiling method with a femtomolar detection limit for metabolic labeling of CGds, allowing for qualitative and quantitative analysis of CGds. The method also enables us to identify the enzyme catalyzing the biosynthesis of cholesteryl 6’-O-acyl glucoside, namely cholesteryl α-D-glucopyranoside 6’-O-acyltransferase (CGAT). Additionally we established the related assays to measure the CGAT activity, as well as the activity of cholesteryl α-D-glucopyranoside 6’-O-phosphatidyltransferase (CGPT). We also characterized CGAT in detail, including pH profile, substrate specificity and kinetic parameters, and its cellular location. CGAT was found to have the following features. (1) It is an outer membrane protein and secreted through the outer membrane vesicle (OMV)-dependent pathway. (2) The enzyme is able to acquire host phospholipids, especially phosphoethanolamine to synthesize CAGs. (3) CGAT has an optimal pH of 4.5, suggesting that CGAT can operate well in the gastric environment and on the epithelial cells. Further studies also showed that CGAT-containing OMVs changed the host cell membrane composition and resulted in the clustering of lipid rafts that enhanced the bacterial adhesion and the T4SS function during the course of infection. The adhesion is associated with the recruitment of integrins α5 and β1 on lipid rafts. Finally, we discovered amiodarone to be a potent inhibitor of CGAT (IC50 = 13.6 μM) that significantly reduced the cell adhesion. | en |
dc.description.provenance | Made available in DSpace on 2021-06-16T09:47:47Z (GMT). No. of bitstreams: 1 ntu-106-D99b46006-1.pdf: 7470918 bytes, checksum: a18e18348bc2b536cf33bf5e690200b5 (MD5) Previous issue date: 2017 | en |
dc.description.tableofcontents | 謝誌 i
Abstract iii 摘要 v Abbreviations vii Content xi List of Figures xv List of Tables xix List of Schemes xx Chapter 1 Introduction 1 1.1 Helicobacter pylori and its infection 1 1.2 Virulence factors 2 1.3 Cholesteryl glucosides 4 1.4 Outer membrane vesicles 7 1.5 Motivations 9 1.5.1 Establishing a method for quantitative and qualitative analysis of CG metabolites 9 1.5.2 Identification and characterization of cholesteryl α-D-glucopyranoside acyltransferase CGAT 10 1.5.3 Functional role of CGAT in H. pylori infection 12 1.5.4 Identification of cholesteryl α-D-glucopyranoside phosphatidyltransferase CGPT 13 Chapter 2 Results and Discussion 15 2.1 Establishing a sensitive method for qualitative and quantitative analysis of cholesteryl α-D-glucopyranoside derivatives 15 2.1.1 MS analysis of native CG derivatives 15 2.1.2 Search of a suitable cholesterol analogue for the purpose of metabolic labeling 19 2.1.3 Construction of the labeling procedure with alkyne probes 26 2.1.4 Identification and quantitation of the CG derivatives 32 2.1.5 Analysis of the composition difference of CG derivatives between H. pylori culture and co-culture with AGS cells 43 2.2 Characterization of CGAT 45 2.2.1 Enzyme identification 45 2.2.2 Cloning and overexpression of recombinant CGAT 51 2.2.3 To Establish the activity assay of CGAT 55 2.2.4 Enzyme kinetics 64 2.2.5 Configurational and substrate specificity 68 2.2.6 Examination of CGAT inhibitors 72 2.2.7 Reversed reaction of CGAT 74 2.2.8 The location of CGAT in H. pylori 76 2.3 The role of cholesteryl α-D-glucopyranoside acyltransferase during infection 78 2.3.1 Translocation of CGAT to host epithelial cells 78 2.3.2 Alternation of lipid composition on gastric epithelial cells after CGAT translocation 83 2.3.3 CGAT produces CAGs which induce lipid raft clustering 86 2.3.4 Enhancement of H. pylori adhesion and CagA translocation 92 2.3.5 Inhibition of CGAT impedes OMV-mediated H. pylori adhesion enhancement 95 2.3.6 Integrin α5β1 involved in lipid raft-induced enhancement of H. pylori adhesion 98 2.4 Identification of cholesteryl α-D-glucopyranoside phosphatidyltransferase 101 2.4.1 Selection and examination of CGPT candidates from H. pylori genome 101 2.4.2. Preliminary investigation of CGPT substrate specificity and assay conditions from H. pylori lysate 105 2.4.3 Trials on purification of CGPT from H. pylori 108 2.4.4 Establishment of a high-throughput screening method for CGPT activity 114 2.4.5 Utilization of transphosphatidylation of phospholipase D in order to synthesize CPGs 117 Chapter 3 Conclusions and Future Perspectives 123 Chapter 4 Materials and Methods 130 4.1 Materials 130 4.2 Experimental methods 139 4.2.1 Analysis methods of CGds 139 4.2.2 Molecular biology and biochemical methods 141 4.2.3 Cell biology methods 149 References 154 Appendix 167 | |
dc.language.iso | en | |
dc.title | 鑑定膽固醇葡萄糖醯基轉移酶及其增強胃幽門螺旋桿菌附著之功能 | zh_TW |
dc.title | Identification of Cholesteryl α-D-Glucoside Acyltransferase in Helicobacter pylori and Its Role to Enhance the Bacterial Adhesion | en |
dc.type | Thesis | |
dc.date.schoolyear | 105-1 | |
dc.description.degree | 博士 | |
dc.contributor.oralexamcommittee | 王雯靜(Wen-Ching Wang),高茂傑(Mou-Chieh Kao),吳東昆(Tung-Kung Wu),史有伶(Yu-Ling Shih) | |
dc.subject.keyword | 胃幽門螺旋桿菌,膽固醇葡萄糖衍生物,外膜囊泡,脂質筏,整合素, | zh_TW |
dc.subject.keyword | Helicobacter pylori,cholesteryl glucoside,HP0499,outer membrane vesicle,lipid raft,integrin, | en |
dc.relation.page | 177 | |
dc.identifier.doi | 10.6342/NTU201700184 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2017-01-23 | |
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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