探討胃幽門螺旋桿菌26695菌株中熱休克蛋白60的位置分布及在不同壓力下對其表現之影響

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研究生：	王維柔 Wang, Wei-Rou
論文名稱：	探討胃幽門螺旋桿菌26695菌株中熱休克蛋白60的位置分布及在不同壓力下對其表現之影響 Studies on the localization of Hsp60 and the effect of various stresses on its expression 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：	49
中文關鍵詞：	胃幽門螺旋桿菌、熱休克蛋白60 、醣基化
外文關鍵詞：	Helicobacter pylori, Heat shock protein 60, protein glycosylation
相關次數：	點閱：2 下載：0
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胃幽門螺旋桿菌（Helicobacter pylori）是一株螺旋狀的微耗氧革蘭氏陰性菌。世界上超過50%人口已被此菌感染，而感染此病原菌會引起人類胃慢性發炎、胃及十二指腸潰瘍，甚至胃癌的危險因子。胃幽門螺旋桿菌具有大量的熱休克蛋白60，而此蛋白也幾乎存在於所有的細菌中。熱休克蛋白60是一種分子伴護蛋白（molecular chaperones），主要存在於細胞質中幫助蛋白質的摺疊及其恆定。然而在近年來的研究中，熱休克蛋白60被發現它可能會出現在細菌的表面，甚至被釋放出細菌外，並且扮演與先前截然不同的角色，例如：幫助細菌感染宿主。胃幽門螺旋桿菌在生長的過程中會持續產生帶有致病因子的外膜囊泡（outer membrane vesicles），這些外膜囊泡可以被宿主吞噬並藉此將致病因子帶到宿主細胞內，因此被認為是細菌感染宿主細胞的另外一種途徑。
在本篇研究中，我們首先證實熱休克蛋白60的確會出現在胃幽門螺桿菌26695菌株外膜的表面，隨後確認他也會被釋放於外膜囊泡，並且以緊密的方式與其結合。當細菌受到不同的環境壓力時常會促使熱休克蛋白的表現，藉由此特性我們探討當胃幽門螺旋桿菌接受不同的壓力時，熱休克蛋白60的表現是否會產生改變。結果指出，在pH值下降的環境中熱休克蛋白60的表現會增加，然而在環境中具有高濃度的氯黴素（Chloramphenicol）則會影響熱休克蛋白60的生成。因為在近年來的研究中發現熱休克蛋白60具有兼差蛋白（moonlighting proteins）的特性，因此我們也比較了胃幽門螺旋桿菌26695野生型菌株與HP0859基因缺失的突變菌株間熱休克蛋白60的差異。在先前研究中我們發現HP0859基因可轉譯成 ADP-L-glycero-D- manno-heptose-6-epimerase，是參與脂多醣（lipopolysaccharide）結構中ADP-L-D-heptose 生合成途徑所需酵素之一，並且進一步發現 HP0859 基因缺失的突變不但會造成絕大部分的inner-core及全部的outer-core及O-antigen缺失，說明此突變菌株會影響到醣基化（Glycosylation）系統的作用。本篇研究結果指出，不管在正常的環境或不同的環境壓力下，當菌熱休克蛋白60位於外膜與內膜時，皆展現了醣基化的可能性，熱休克蛋白質60醣基化修飾的生理意義仍待後續研究來發展。

Helicobacter pylori is a Gram-negative, microaerophilic, spiral-shaped and flagellated bacterium. Approximately half of the world’s population was infected by H. pylori, which has been considered as a risk factor for inducing chronic gastritis, peptic ulcer and gastric cancer. Heat shock protein 60 (Hsp60) , one of the most abundant proteins in H. pylori, is found in nearly all bacterial species with a primary function in the cytoplasm to assist protein folding and maintain cell proteostasis. However, recent data indicate that Hsp60 may play an important role in bacterial infections when it is attached to the cell surface or secreted outside the cell. H. pylori can produce outer membrane vesicles (OMVs) that have been demonstrated to contain several adhesins, virulence factors and LPS. Accumulated finding suggest that OMVs might play a central role in H. pylori pathogenesis by acting as delivery vehicles to transport virulence factors.
In this study, we confirmed the possible exposure of Hsp60 on the surface of H. pylori 26695 strain. We also isolated OMVs from H. pylori to investigate the possible presence of Hsp60. According to the obtained results, Hsp60 can be released into OMVs and tightly associated with OMVs. Due to the conventional concept that the synthesis of heat shock proteins is usually promoted in response to various environmental stresses, we tried to gain deeper insight into the expression of H. pylori Hsp60 under different stress conditions. The results show that the expression of H. pylori Hsp60 is affected by some stress-treating conditions such as pH decreasing and chloramphenicol treatment. Moreover, because of the possible moonlighting properties of Hsp60, we compared Hsp60 from H. pylori 26695 wild-type strain with that from the HP0859 knockout mutant which is lacking most of the inner-core, and the entire the outer-core and O-antigen, and has been demonstrated previously in our laboratory to fall short of a protein glycosylation system, to investigate the possibility of protein glycosylation under different stress conditions. The results indicate that Hsp60 has a high possibility of protein glycosylation under normal and various stress conditions in H. pylori. The biological significance of Hsp60 glycosylation remains to be explored in the future.

中文摘要    i
Abstract    ii
Table of Content    iv
List of Tables    vi
List of Figures    vii
Abbreviations    viii
Chapter 1 Introduction    1
1.    The history and microbiology of Helicobacter pylori    1
2.    The pathogenesis of H. pylori infection    2
3.    Lipopolysaccharide (LPS) of H. pylori    3
4.    The characteristics and moonlighting actions of heat shock protein 60 (Hsp60)    5
5.    Outer membrane vesicles (OMVs)    6
6.    Motivation of this study    7
Chapter 2 Materials and methods    9
1.    Bacterial strains and growth conditions    9
2.    Isolation of outer membrane vesicles (OMVs)    9
3.    Total cell lysate extraction    10
4.    The colony blot of H. pylori    10
5.    Confocal microscopy    11
6.    Dissociation assay    11
7.    Protease and trypsin digestion of outer membrane vesicles    12
8.    The expression of Hsp60 under different stress conditions    12
9.    Cell fractionation    13
10.    SDS-PAGE and Western immunoblotting    13
11.    Cell culture of gastric cells, H. pylori infection and OMV treatment    14
12.    Bioinformatic analyses    15
13.    Quantification of Western blot band intensities and statistical analysis    15
Chapter 3 Results    16
1.    Sequence analysis of Hsp60 protein in H. pylori 26695 strain    16
2.    Surface localization of H. pylori Hsp60    16
3.    The association of Hsp60 with outer membrane vesicles of H. pylori    17
4.    The effect of bacterial growth time on Hsp60 expression    18
5.    The effect of different stress conditions on Hsp60 expression    18
6.    The statuses of protein glycosylation of H. pylori Hsp60 under different stress conditions    19
7.    The translocation of Hsp60 into AGS cells after co-culturing with H. pylori or OMVs    22
Chapter 4 Discussion    23
Chapter 5 References    27
List of Tables    31
Table 1. Bacterial strains used in this study.    31
Table 2. Sequence comparison of H. pylori Hsp60 protein with homologues from other Gram-negative bacteria.    32
Figures    33
Figure 1. The bioinformation of Hsp60 in H. pylori 26695 strain.    33
Figure 2. Multiple sequence alignment of H. pylori Hsp60 protein with their homologues from other bacterial species.    35
Figure 3. Surface location of H. pylori Hsp60.    37
Figure 4. The association of Hsp60 with OMVs of H. pylori.    38
Figure 5. The effect of bacterial growth time on Hsp60 expression.    40
Figure 6. The effect of different stress conditions on Hsp60 expression.    44
Figure 7. The statuses of protein glycosylation of H. pylori Hsp60 under different stress conditions.    48
Figure 8. The translocation of Hsp60 into AGS cells after co-culturing with H. pylori or OMVs.    49

                                

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