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研究生： 郭承儒
Kuo, Cheng-Ju
論文名稱： 全基因體學鑑定出血性大腸桿菌於感染線蟲所需毒理因子之相關基因
Genome-Wide Identification of Enterohemorrhagic E. coli Virulence-Related Genes in Caenorhabditis elegans
指導教授： 陳昌熙
Chen, Chang-Shi
學位類別： 博士
Doctor
系所名稱： 醫學院 - 基礎醫學研究所
Institute of Basic Medical Sciences
論文出版年： 2018
畢業學年度： 106
語文別： 英文
論文頁數： 60
中文關鍵詞： 出血性大腸桿菌 、RfaD/GmhD/WaaD 、脂多醣 、抗微生物胜肽 、腸道先天免疫 、秀麗隱桿線蟲
外文關鍵詞： enterohemorrhagic Escherichia coli (EHEC), RfaD/GmhD/WaaD, lipopolysaccharide (LPS), antimicrobial peptides (AMPs), intestinal innate immunity, Caenorhabditis elegans
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    • 出血性大腸桿菌 O157:H7 是藉由食物傳播且難以治療的致病菌，會在全世界造成
    人類嚴重疾病。現今，對於出血性大腸桿菌並無專一性療法，使用傳統抗生素的治療方法極具爭議與限制。因此，迫切需要研發對出血性大腸桿菌於控制和治療之新穎方法。脂多醣，其位於出血性大腸桿菌細胞外膜外層之醣脂質。藉由全基因體篩選，我們發現參與出血性大腸桿菌脂多醣核心合成的基因，rfaD，在活體實驗中對出血性大腸桿菌之致病力扮演極重要角色。破壞腸道出血性大腸桿菌脂多醣合成基因rfaD 後，除使出血性大腸桿菌對線蟲造成減毒外，亦減少其對線蟲和小鼠寄殖於腸道之能力，且出血性大腸桿菌需要rfaD 基因來抵禦抗微生物胜肽和宿主腸道之免疫系統。值得注意的是，破壞rfaD 基因不會影響到出血性大腸桿菌的生長，因此以rfaD 基因作為治療標的，並不會使出血性大腸桿菌產生抗藥性。我們於活體實驗中之結果顯示，破壞出血性大腸桿菌rfaD 基因後，細菌對宿主腸道先天免疫系統極具敏感性。這暗示了或許以RfaD 蛋白或是脂多醣核心合成路徑之蛋白為標的，可提供治療出血性大腸桿菌感染非抗生素且新穎之療法。

    Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is one of the most challenging foodborne pathogens causing severe diseases in humans worldwide. Currently, there is no specific treatment available for EHEC infection and the use of conventional antibiotics is contraindicated. Therefore, identification of potential therapeutic targets and development of effective measures to control and treat EHEC infection are needed. Lipopolysaccharides (LPS) are surface glycolipids found on the outer membrane of gram-negative bacteria, including EHEC. We unveiled that the EHEC rfaD gene that functions in the biosynthesis of the LPS core plays important roles in pathogenesis in vivo through an unbiased genome-wide screening. Disruption of the EHEC rfaD gene confers attenuated toxicity in C. elegans and less bacterial colonization in C. elegans and mouse models. Moreover, the rfaD gene is also required for EHEC defense against antimicrobial peptides and host intestinal immunity. It is worth noting that rfaD mutation did not interfere with the growth kinetics when compared to the wild-type EHEC cells. Targeting rfaD as a therapeutic target does not confer resistance of EHEC to antibiotic. Taken together, we demonstrated that mutations of the EHEC rfaD gene confer hypersusceptibility to host intestinal innate immunity in vivo, and suggest that targeting the RfaD or the core LPS synthesis pathway may provide novel non-antibiotic regimens for EHEC infection.

    中文摘要 III Abstract IV 誌謝 V Introduction 1 Materials and methods 4 Bacterial and C. elegans strains 4 Construction of the plasmid 6 Construction of the Enterohemorrhagic E. coli O157:H7 mutants 7 Survival analysis 7 LPS extraction and visualization 8 Bacterial colonization 9 Microvillar ACT-5 cellular localization 10 Serum killing assay 10 In vivo and ex vivo imaging analysis of EHEC infected mice 10 Susceptibility to antimicrobial peptides 11 Results 12 Functional genomic screening of the EHEC EDL933 Tn5 transposon library for mutants with decreased virulence in C. elegans 12 Disruption of the rfaD gene decreases toxicity of EHEC O157:H7 to C. elegans 13 Disruption of the core LPS Biosynthesis genes decrease toxicity of EHEC O157:H7 to C. elegans
 14 Mutation in rfaD confers reduced bacterial colonization and less microvillar actin rearrangement in the intestine of C. elegans 16 Disruption of rfaD gene reduces bacterial colonization in the intestine of mouse 18 Deletion of rfaD increases the susceptibility of EHEC to human serum killing 19 Disruption of the rfaD gene increase the susceptibility of EHEC to AMPs in vitro 20 Deletion of rfaD Confers the Hypersusceptibility of EHEC to Host Intestinal Immunity In vivo 21 Discussion 22 References 27 Figures 36 Figure 1. Lipopolysaccharide (LPS) structure and the core-lipid A biosynthetic pathway. 36 Figure 2. Screening of the EDL933 transposome mutant library. 37 Figure 3. EHEC strain EDL933 EZ-Tn5 transposon library screening for the attenuated toxicity mutants in C. elegans. 38 Figure 4. Disruption of rfaD gene and the genes involved in EHEC core LPS biosynthesis confers attenuated toxicity. 39 Figure 5. Survival curves of N2 animals fed with E. coli OP50 and OP50:rfaD. 41 Figure 6. Deletion of rfaD attenuates the toxicity of E. coli O157:H7 strain HER1266 in C. elegans. 42 Figure 7. Disruptions of waaI and wbdP confer attenuated toxicity of EDL933 in C. elegans. 43 Figure 8. Mutation in rfaD gene reduces bacterial colonization in C. elegans. 44 Figure 9. Mutation in rfaD reduce EHEC-induced intestinal microvillar actin rearrangement in C. elegans. 45 Figure 10. Disruption of rfaD gene reduces bacterial colonization in the intestines of mice. 46 Figure 11. Deletion of EHEC rfaD increases its susceptibility to human serum killing and the antimicrobial peptide LL-37. 48 Figure 12. The rfaD mutation increases the susceptibility of EHEC to host intestinal innate immunity. 50 Figure 13. Proposed model of EHEC infects C. elegans and host intestinal antimicrobial peptides target EHEC in the absence of intact LPS. 51 Table 52 Table 1: Bacteria strains and plasmids used in this study 52 Table 2: The nematode strains used in this study. 53 Table 3: Transposon candidate genes list………….………………………………………………….………………..54 Table 4: Minimum inhibitory concentration of Polymyxin B and Colistin 56 Appendix 57

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