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研究生： 陳姵㚬
Chen, Pei-Chun
論文名稱： HP0858及HP0279基因破壞對於胃幽門螺旋桿菌26695菌株脂多醣結構/功能及蛋白質醣化的影響
The effect of HP0858 and HP0279 gene disruption on the structure/function of lipopolysaccharide and protein glycosylation in Helicobacter pylori 26695 strain
指導教授： 高茂傑
Kao, Mou-Chieh
口試委員: 藍忠昱
Lan, Chung-Yu
殷献生
Yin, Hsien-Sheng
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： 2020
畢業學年度： 108
語文別： 英文
論文頁數： 52
中文關鍵詞： 胃幽門螺旋桿菌 、脂多醣 、庚醣 、HP0858基因 、HP0279基因
外文關鍵詞： Helicobacter pylori, lipopolysaccharide, heptose, HP0858 gene, HP0279 gene
相關次數： 點閱：2  下載：0 
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    • 胃幽門螺旋桿菌 (Helicobacter pylori)是一種螺旋狀的革蘭氏陰性菌，它寄生於哺乳類動物的胃黏膜細胞，全球約有50%的人口被胃幽門螺旋桿菌感染，研究已經發現感染此菌會導致慢性胃炎、胃部或十二指腸潰瘍，且伴隨感染時間增長等因素會增加宿主罹患胃腺癌甚至是黏膜相關淋巴組織淋巴瘤的風險。了解胃幽門螺旋桿菌感染的致病機制有助於治療方法研究的開發。脂多醣 (lipopolysaccharide, LPS)是胃幽門螺旋桿菌的致病因子之一。脂多醣的結構主要有三部分：脂質A (Lipid A)、核心寡糖 (Core oligosaccharide)及O抗原 (O-antigen)，其中核心寡糖的主要成分之一是庚醣 (Heptose)。最近有多篇研究指出庚醣生合成反應的一中間產物ADP heptose是新被發現之病原相關分子樣式，其會導致宿主胃壁上皮細胞中介白素-8 (Interleukin-8)大量活化。在這篇報告中，我們成功建構了HP0858基因被破壞的突變株，再加上實驗室先前已建構的和庚糖生合成途徑相關 (HP0857、HP0859、HP0860)及庚糖轉移酶I (HP0279)基因被破壞的突變株，讓我們有機會評估了胃幽門螺桿菌野生型26695菌株和這些與庚糖生合成相關的基因破壞突變株之間的差異。我們發現這些基因的突變會導致截斷的脂多醣結構，並增加胃幽門螺旋桿菌對清潔劑SDS及抗生素novobiocin的敏感性，最重要的是這些突變似乎也降低了胃幽門桿菌的致病性。我們希望這些發現能為尋找新的針對胃幽門螺桿菌感染的抗生素或疫苗開發的靶標提供啟示。

    Helicobacter pylori is a spiral-shaped Gram-negative bacterium that is parasitic to the gastric mucosal cells of mammals. About 50% of the world's population is infected with H. pylori. It has been reported that H. pylori infection results in chronic gastritis, gastric or duodenal ulcers, and gradually increases the risk for development of gastric adenocarcinoma and mucosa-associated lymphoid tissue lymphoma. Understanding the pathogenic mechanism of H. pylori infection will help the development of therapeutic approaches. Lipopolysaccharide (LPS) is one of the pathogenic factors of H. pylori. The composition of LPS can be dived into three parts: Lipid A, core oligosac-charide and O-antigen. One of the main components of core oligosaccharides is heptose. Recently, several studies have pointed out that one of the intermediates of the heptose synthetic pathway, ADP heptose, functions as a novel pathogen-associated molecular pattern and can promote interleukin-8 activation in gastric epithelial cells. In this report, we constructed HP0858 gene disruption mutant. Along with other gene disruption mutants in the heptose biosynthetic pathway (including HP0857, HP0859 and HP0860) and heptosyltransferase I (encoded by HP0279) generated by other students in our laboratory previously, we had the chance to evaluate the differences of various physiological functions among H. pylori wild-type strain 26695 and these heptose biosynthesis-related isogenic mutants. We revealed the mutations in the heptose biosynthetic pathway-related genes and the gene encoded heptosyltransferase I could result in a truncated LPS structure. These mutations also increase the bacterial sensitivity to detergents and antibiotics. More importantly, these mutations reduce the bacterial infectivity of H. pylori. We hope that these findings will provide inspiration for finding new targets for the development of new antibiotics or vaccines against H. pylori infection.

    中文摘要 i ABSTRACT ii TABLE of CONTENTs iii ABBREVIATIONs v INTRODUCTION 1 Historical background of Helicobacter pylori 1 Determinants of H. pylori pathogenesis 1 Lipopolysaccharide (LPS) 2 Heptose biosynthetic pathway 3 Outer membrane vesicles (OMVs) 4 The motivation of this study 5 MATERIALs and METHODs 6 Bacterial strains and generation of mutants 6 Bioinformatics analysis 7 Bacterial liquid culture and total cell lysate preparation 7 LPS analysis by silver stain and immunoblotting 8 Bacterial growth curves analysis 9 SDS and novobiocin sensitivity analysis 9 Hydrophobicity and autoaggregation assays 9 Isolation of OMVs 10 Analyses of bacteria and bacterial OMVs for virulence factors 10 Cell culture and infection experiments 10 IL-8 measurements 11 Cell fractionation and immunoblotting 11 Statistical analysis 12 RESULTs 13 Sequence analysis of HP0858 and HP0279 in H. pylori 26695 strain 13 Gene disruption in HP0858 or HP0279 gene results in a truncated LPS structure 13 Defects in the heptose biosynthetic pathway of LPS have only a slight effect on the growth of H. pylori 15 Defects in the LPS inner core exhibit hypersensitivity to SDS and novobiocin 15 Defects in the LPS inner core increased surface hydrophobicity in H. pylori 16 AlpB may be glycosylated and the glycosylation process may shares enzymes from the LPS biosynthetic pathway 16 Mutations on the LPS inner core reduce the bacterial infectivity of H. pylori 17 Mutations on the biosynthesis of LPS inner core decreased the amount of key virulence factors present in OMVs 18 ADP heptose triggers the activation of IL-8 in AGS cells after H. pylori infection 18 DISCUSSION 20 REFERENCEs 23 TABLEs 27 FIGUREs 30 SUPPLEMENTARY DATA 49

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