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系統識別號 U0026-0608201223045500
論文名稱(中文) 以功能性基因體學法分析出血性大腸桿菌於線蟲中所需之毒理因子
論文名稱(英文) Functional Genomic Analysis of the Virulence of Enterohemorrhagic Escherichia coli 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) 周廷蓁
研究生(英文) Ting-Chen Chou
學號 S16994108
學位類別 碩士
語文別 英文
論文頁數 82頁
口試委員 口試委員-何漣漪
口試委員-鄧景浩
口試委員-邱浩傑
口試委員-蕭崇瑋
指導教授-陳昌熙
中文關鍵字 出血性大腸桿菌  脂多醣  線蟲 
英文關鍵字 Enterohemorrhagic Escherichia coli (EHEC)  lipopolysaccharide (LPS)  Caenorhabditis elegans 
學科別分類
中文摘要 出血性大腸桿菌(Enterohemorrhagic E. coli,簡稱EHEC),主要由血清型0157:H7之大腸桿菌引起,為人畜共通病原菌。出血性大腸桿菌的感染主要是食物污染引起,特別是受牛隻排泄物污染。出血性大腸桿菌感染在臨床上的病徵從一般的腹瀉至出血性腹瀉都可能發生,十歲以下的孩童及免疫力差的老人受感染後易併發溶血性尿毒症候群(hemolytic uremic syndrome;HUS)或栓塞性血小板減少性紫斑症(thrombotic thrombocytopenic purp-ura;TTP)等併發症。目前認為典型的EHEC至少具有三大類重要致病因子:類志賀毒素(Shiga-like toxins, Stx)、腸道細胞消除基因組(locus of enterocyte effacement, LEE),及含有溶血基因的質體pO157。過去針對治療策略上,科學家積極研發抗生素和疫苗來對抗此傳染病,但截至目前為止,效果並不彰。因此,目前有效的去預防及控制EHEC的感染是極為重要的。此外,近年來模式生物線蟲已被廣泛的用來研究不同種類的致病菌之致病機轉,因此,本實驗室利用這種在其自然棲息地就可能接觸到出血性大腸桿菌的線蟲,來作為模式宿主並且探討出血性大腸桿菌的致病機轉。
由實驗結果發現,在過去已知對於哺乳類細胞具有毒性的第一型類志賀毒素(Shiga-like toxin 1, Stx1)也參與對線蟲的感染。此外,我們也將過去在動物細胞模式中找到的毒理因子進行測試,卻發現這些特定出血性大腸桿菌基因的缺失對其毒殺線蟲並無太大的影響。這暗示了可能有其它未被發掘的細菌因子參與在線蟲的感染,因此,我們建立了出血性大腸桿菌的跳躍基因子突變基因庫,來篩選出主要參與出血性大腸桿菌毒殺線蟲的的毒理因子。結果顯示,參與出血性大腸桿菌之脂多醣(Lipopolysaccharide)合成的許多基因,對於出血性大腸桿菌在線蟲中的完整毒性是必須的,也發現出血性大腸桿菌的脂多醣和線蟲正常食物來源E.coli OP50之脂多醣組成有所不同。在此,我們推測出血性大腸桿菌的脂多糖是影響線蟲壽命長短的關鍵因素,同樣的我們也發現藉由這樣基因篩選所找到的出血性大腸桿菌突變株對人類結腸細胞的毒性也顯著的降低了。藉此,我們希望利用此簡單的多細胞真核生物─線蟲,找出未知或新穎的出血性大腸桿菌毒理因子,也許未來將可提供有別於過去傳統的治療方針。
英文摘要 Enterohemorrhagic Escherichia coli (EHEC) serotype O157:H7 is a major foodborne pathogen causing severe disease in humans worldwide. Three major groups of virulence factors of E. coli O157:H7 have been identified including the Shiga-like toxins, the products of the pathogenicity island called the locus of enterocyte effacement (LEE), and the products of the F-like plasmid pO157. Several strategies for therapy have been studied including the use of antibiotics and vaccination. However, there is no specific treatment for E. coli O157:H7 infection so far and the use of antibiotics may be contraindicated. Therefore, highly effective measures for prevention and control of E. coli O157:H7 infection are essential. Recent reports suggested that the nematode Caenorhabditis elegans can be used as an infection model for diverse groups of bacterial pathogens. Here, we illustrated the C. elegans, which may encounter EHEC in its habitat naturally, as a model host for studying EHEC infection.
Our current data suggested that the Shiga-like toxin 1 (Stx1), an important toxin virulence factor for mammalian cells, was required partly for the virulence of E. coli O157:H7 to C. elegans. Moreover, we analyzed the roles of the other well known EHEC virulence factors, which are identified in mammalian cell culture systems, in C. elegans. Finally, we have established a genome-wide transposon mutagenesis library to screen for the bacterial factors that are required for the pathogenesis of EHEC infection in C. elegans. From this genetic screen, we have discovered that the genes involved in lipopolysaccharide (LPS) synthesis may play important roles on the pathogenesis of O157:H7. We also found the component of EHEC LPS is different from that of E.coli OP50. This may suggest that the LPS of EHEC is a key factor on influencing the lifespan of C. elegans. We envision this simple, yet elegant, in vivo system will be useful for identifying previously uncharacterized EHEC virulence factors, and may pave a way for novel treatments beyond traditional therapy.
論文目次 中文摘要…………………………………………………………………1
Abstract…………………………………………………………….........3
Introduction……………………………………………………………..7
Materials and Methods………………………………………………..10
Results…………………………………………………………………..21
E. coli O157:H7 intoxicates and kills C. elegans........21
E. coli O157:H7 intoxicates C. elegans, in part, by Shiga-like toxin 1 (Stx1)......22
Other known bacterial factors virulent for mammalians are not all involved in C. elegans infection………25
Screening of the E. coli EDL933 EZ-Tn5 transposon library for mutants with decreased virulence in C. elegans………………26
Transposon insertions in several LPS synthesis genes confer attenuated toxicity in C. elegans……………………………27
Disruptions of the rfaD gene alter the LPS structure and result in decreased toxicity of E. coli EDL933…………………………28
Mutation in rfaD resulted in reduced bacterial colonization ability and host ACT-5 ectopic expression in the intestine of C. elegan..30
rfaD mutation reduces severe bag of worms (Bag) phenotype induced by the EDL933 wild type……………………31
LPS may be a virulence factor of EDL933 for C.elegans…....32
rfaD mutation also results in the reducd toxicity to mammalian cells…….33
Tesst of a small molecule targets on RfaD as an EHEC therapeutics in C. elegans………………………………………….33
Conclusion and Discussion…………………………………………….35
References………………………………………………………………40
Tables………………………………………………………………44
Figures……………53
Supplemental Data…………………………………………………….70
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