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系統識別號 U0026-1908201223300200
論文名稱(中文) 參與出血性大腸桿菌寄殖之線蟲宿主基因之正向遺傳學篩選
論文名稱(英文) A forward genetic screen for host factors involved in enterohemorrhagic E. coli colonization in Caenorhabditis elegans
校院名稱 成功大學
系所名稱(中) 生物化學暨分子生物學研究所
系所名稱(英) Department of Biochemistry and Molecular Biology
學年度 100
學期 2
出版年 101
研究生(中文) 郭承儒
研究生(英文) Cheng-Ju Kuo
學號 S16991029
學位類別 碩士
語文別 英文
論文頁數 72頁
口試委員 口試委員-鄭宏祺
口試委員-何漣漪
口試委員-曾炳輝
口試委員-鄧景浩
指導教授-陳昌熙
中文關鍵字 秀麗隱桿線蟲  出血性大腸桿菌  粘著/塗抹病變 
英文關鍵字 C. elegans  Enterohaemorrhagic E. coli (EHEC)  Attaching and effacing (A/E) lesion 
學科別分類
中文摘要 中文摘要
大腸桿菌O157:H7是一種會隨著食物或水源傳播感染的出血性大腸桿菌,此致病菌會導致人們腹瀉,出血性結腸炎,溶血性尿毒症甚至腎衰竭等症狀。現今,對於出血性大腸桿菌感染之治療方式仍限制於給予支持性照護來減輕症狀及併發症的預防。目前科學家們已經利用許多模式動物來進行出血性大腸桿菌的研究,然而,在自然情況下感染出血大腸桿菌且能夠進行遺傳操作之模式生物的缺乏下,阻礙了出血性大腸桿菌感染於活體內遺傳方法之整體性研究。在此,我們利用了自然環境下會被出血性大腸桿菌感染的可遺傳操作生物—秀麗隱桿線蟲(Caenorhabditis elegans),來研究宿主對出血性大腸桿菌的先天免疫反應。
我們發現出血性大腸桿菌會感染並殺死線蟲,也發現相同於此致病菌感染哺乳動物之特徵如細菌寄殖於腸道中且引發粘著/塗抹病變(A/E lesions)現象,在此病菌感染的線蟲腸道中亦會出現。兩種演化高保留度之先天免疫的訊息路徑,p38 MAP kinase 和DAF-2 insulin-like的訊息路徑,在線蟲遭受出血大腸桿菌感染時也會被活化並調控宿主對此病菌的感受性。我們利用甲基磺酸乙酯(EMS)這種突變劑去篩選對於出血性大腸桿菌寄殖於宿主所參與的宿主因子。在所篩選的124株線蟲突變株當中,其中一株突變株,wf059,發現其對於出血大腸桿菌是有抗性的,並且經遺傳學方法分析,得知其突變區可能位於染色體I上。既然先天免疫在許多生物體中是相當保守性的反應,我們希望藉由瞭解線蟲對於出血性大腸桿菌感染的免疫防禦系統之分子基礎,可為免疫系統抵禦出血性大腸桿菌之研究,帶來新的曙光。
英文摘要 Abstract
Escherichia coli O157:H7 is a member of pathogenic E. coli, known as enterohemorrhaghic E. coli (EHEC), can colonize the gastrointestinal tract and cause life-threatening infections worldwide. However, current treatment for the EHEC infection is still limited to supportive care. Although several animal models have been proposed for studying the EHEC infection, the lack of naturally infected and genetic tractable animal model hinders the study of EHEC infection with holistic and unbiased genetic screening approaches in vivo. Here we applied the genetic tractable animal, Caenorhabditis elegans—which may encounter EHEC in its niche naturally as a surrogate host for studying the host innate immunity to this pathogen.
Our results showed that E. coli O157:H7, infected and killed C. elegans. Colonization and induction of the characteristic attaching and effacing (A/E) lesions by E. coli O157:H7 were concomitantly demonstrated in the intact intestinal epithelium of C. elegans in vivo. The C. elegans p38 MAP kinase and the DAF-2 insulin-like signaling pathways, two evolutionally conserved innate immune signaling pathways, were activated and mediated in the regulation of host susceptibility to EHEC infection. We also applied the EMS-mediated genetic screen for the host factors involved in E. coli O157:H7 colonization in C. elegans. Among the 124 candidates, one of the allele, wf059, with a mutation been mapped to the chromosome I, showed resistant to E. coli O157:H7.Given that the innate immune responses are highly conserved among different organisms, understanding the molecular basis of the immune defense systems in C. elegans should shed light onto some aspects of immunity against EHEC infection.
論文目次 Contents
誌謝 1
中文摘要 2
Abstract 3
Introduction 4
Materials and Methods 7
Bacterial and Nematode Strains 7
E. coli O157:H7 Killing Assays 8
Colony Forming Units (CFU) 10
C. elegans Images 11
Western Blot 13
Genetic screen for mutants with enhanced EHEC colonization in the intestine 14
SNP mapping 15
General Stressors Analysis 15
Results 16
E. coli O157:H7 intoxicates and kills C. elegans 16
E. coli O157:H7 colonizes and induces A/E lesions in C. elegans 18
E. coli O157:H7 induces severe bag of worms (Bag) phenotype in C. elegans 22
The p38/MAPK and DAF-2 insulin-like signal pathways are activated and regulate the susceptibility of C. elegans to E. coli O157:H7 25
The p38/MAPK and DAF-2 insulin-like signal pathways function autonomously in the intestinal cells of C. elegans for E. coli O157:H7 defense 28
Genetic Screen for C. elegans mutants with enhanced colonization in intestine 29
YQ032 is resistant to E. coli O157:H7 30
SNP mapping of wf059 33
wf059 is resistant specifically to E. coli O157:H7 infection 34
Discussion 36
Reference 38
Table 43
Table 1. Bacterial and nematode strains and plasmids used for this study 43
Figures 45
Figure 1 45
Figure 2 48
Figure 3 51
Figure 4 53
Figure 5 56
Figure 6 60
Figure 7 61
Figure 8 62
Figure 9 64
Figure 10 67
Figure 11 72
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