胃幽門桿菌HP0860基因缺失對脂多醣生合成的影響

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研究生：	邱淑芬 Chiu, Sue-Fen
論文名稱：	胃幽門桿菌HP0860基因缺失對脂多醣生合成的影響 Effects of HP0860 knockout mutations on lipopolysaccharide biosynthesis in Helicobacter pylori
指導教授：	高茂傑 Kao, Mou-Chieh
口試委員:	高茂傑 Kao, Mou-Chieh 張晃猷 Chang, Hwan-You 彭慧玲 Peng, Hwei-Ling
學位類別：	碩士 Master
系所名稱：	生命科學暨醫學院 - 分子醫學研究所 Institute of Molecular Medicine
論文出版年：	2012
畢業學年度：	100
語文別：	英文
論文頁數：	56
中文關鍵詞：	胃幽門螺旋桿菌、脂多醣、HP0860
外文關鍵詞：	Lipopolysaccharwide
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胃幽門桿菌為一螺旋狀的革蘭氏陰性微耗氧菌，主要寄居在胃黏膜的上皮細胞，是一個引起胃潰瘍、胃癌與胃黏膜相關淋巴癌的危險因子。在眾多胃幽門桿菌的致病因子中，脂多醣 (lipopolysaccharide)是革蘭氏陰性菌細胞膜主要成分之一，除了維持外膜構造上的穩定外而且會誘發宿主產生強烈免疫反應。脂多醣在結構上分成三個部分包含脂質A (lipid A) 、核寡糖 (core oligosaccharide) 和O抗原 (O-antigen)。研究顯示在大腸桿菌中，當脂多醣內核寡糖的主要成分ADP-L-D-heptose若消失，會造成脂多醣的嚴重缺損並降低菌株的致病力。而根據生物資訊比對結果，胃幽門桿菌的 HP0860基因被預測可轉譯成D-D-Heptose-1,7-bisphosphate phosphatase (GmhB)，為參與ADP-L-D-heptose生合成途徑所需酵素之一。因此，本篇論文主要在於分析HP0860基因產物的特性，進而探討HP0860在脂多醣內核寡糖生合成所扮演的角色。實驗結果顯示HP0860在自然環境下可能以單體存在，而酵素活性測定也證實HP0860具有將D-glycero-D-manno-heptose-1,7-bisphosphate 轉化為D-glycero-D-manno-heptose
-1-phosphate 的催化活性 (kcat/Km = 2188 mM-1s-1)。另外，我們建構了HP0860基因缺失的菌株，並比較其與正常野生菌株相關生理特徵之差異。結果顯示HP0860基因缺失的菌株除了產生具有缺陷的脂多醣結構，同時也生成了部份完整的脂多醣結構，我們推測在胃幽門桿菌中可能存在具有和HP0860功能相似的蛋白補償HP0860基因的缺失。此外，當HP0860基因缺失時，菌體的生長速率、貼附能力以及受感染的胃腺癌細胞(AGS cells) 呈現典型蜂鳥狀型態 (hummingbird) 的現象皆受到影響。值得注意的是，我們也發現HP0860基因缺失所造成的脂多醣結構改變，會間接影響外膜囊泡 (outer membrane vesicle) 內蛋白質的組成成分。綜合以上的結果，本研究除了分析HP0860蛋白的特性及功能，同時也證實HP0860確實參與胃幽門桿菌脂多醣內核寡糖生合成，對未來治療及防範幽門桿菌感染的策略提供新的概念。

Helicobacter pylori, a Gram-negative, microaerophilic spiral bacterium that colonizes the mucosa of human stomach, has been considered a risk factor for human gastric diseases. Lipopolysaccharwide (LPS), one of the virulence factors in H. pylori, is composed of lipid A, core oligosaccharide and O-antigen polysaccharide, and is thought to be toxic with potent immunomodulating and immunostimulating properties. In the current study, the HP0860 gene from H. pylori, which was predicted to encode the D-D-Heptose-1,7-bisphosphate phosphatase (GmhB) involved in the synthesis of ADP-L-D-heptose for the assembly of LPS inner core, was cloned and characterized. We reported HP0860 protein under native conditions is monomeric in solution, and confirmed HP0860 functions as a phosphatase by converting D-glycero-D-manno
-heptose-1,7-bisphosphate into D-glycero-D-manno-heptose-1-phosphate (kcat/Km = 2188 mM-1s-1). Subsequently, we constructed an HP0860 knockout mutant and examined its phenotypic properties. The HP0860 knockout mutant contained both mature and immature forms of LPS structure, suggesting another protein present in the HP0860 knockout mutant might be able to partially compensate for the loss of HP0860 activity. In addition, we also revealed that the HP0860 knockout mutant exhibited a decreased growth rate, less classic hummingbird phenotype showing by the infected AGS cells and a lower adherence on H. pylori-infected AGS cells, suggesting that H. pylori lacking HP0860 appeared to be less virulent. Furthermore, we provided the evidence that mutation of the genes involved in LPS biosynthesis altered the sorting of cargo proteins into outer membrane vesicles (OMVs). In conclusion, the findings in this report confirmed the involvement of HP0860 in the LPS inner core biosynthesis and added a new understanding for future developing novel antimicrobial agents against H. pylori infection.

中文摘要    I
Abstract    II
致謝    III
Table of contents    IV
List of tables    VI
List of figures    VII
Chapter 1 Introduction    1
Chapter 2 Materials and methods    8
2.1 Materials    8
2.2 Bacterial strains, plasmids and cell culture    8
2.3 Molecular cloning of HP0860 gene    9
2.4 HP0860 protein expression and purification    10
2.5 Protein SDS–PAGE and immuno-blotting analysis    10
2.6 Determination of the oligomeric state of HP0860 protein    11
2.7 Circular dichroism analysis of the secondary structure of HP0860 protein    12
2.8 Kinetic analysis of HP0860 protein    12
2.9 Construction of HP0860 knockout mutant and complementary mutant    14
2.10 Growth curve analysis of H. pylori    16
2.11 Isolation and phenotypic analysis of H. pylori LPS    16
2.12 The analysis of morphological changes on H. pylori-infected AGS cells    17
2.13 Imaging of H. pylori-infected AGS cells with scanning electron microscopy    18
2.14 Isolation and analysis of H. pylori outer membrane vesicles (OMVs)    19
Chapter 3 Results    20
3.1 Sequence analysis of HP0860 protein in H. pylori 26695    20
3.2 The cloning of HP0860 gene and expression of HP0860    21
3.3 The oligomeric state of HP0860 protein in solution    22
3.4 Circular dichroism analysis of the secondary structure of HP0860 protein    22
3.5 Steady state kinetic analysis of HP0860    23
3.6 Confirmation of HP0860 knockout mutant and complementary mutant    24
3.7 Effects of HP0860 mutations on H. pylori growth curve    25
3.8 Effects of HP0860 mutations on LPS expression    25
3.9 Effects of HP0860 mutations on protein contents of outer membrane vesicles    26
3.10 Effects of HP0860 mutations on AGS cells morphological changes    27
3.11 Morphological analysis of H. pylori attached to AGS cells using SEM    27
Chapter 4 Discussions    29
Chapter 5 References    33
Table    39
Figure    43
                                

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