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研究生： 謝思萱
Xie, Si-Xuan
論文名稱： 原噬菌體φNCKUH-21在艱難梭菌致病機轉中的角色
The role of prophage φNCKUH-21 in Clostridium difficile pathogenesis
指導教授： 陳振暐
Chen, Jenn-Wei
學位類別： 碩士
Master
系所名稱： 醫學院 - 微生物及免疫學研究所
Department of Microbiology & Immunology
論文出版年： 2019
畢業學年度： 107
語文別： 英文
論文頁數： 65
中文關鍵詞： 艱難梭菌 、原噬菌體 、CI抑制因子 、致病機制
外文關鍵詞： Clostridium difficile, Prophage, CI repressor, Pathogenesis
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    • 艱難梭菌是革蘭氏陽性、厭氧、會形成孢子的病原體，細菌感染可引起嚴重的腸道疾病，包括抗生素相關性腹瀉，中毒性巨腸症和偽膜性結腸炎。在先前的文獻中，我們在台灣艱難梭菌核醣體027臨床分離株NCKUH-21的全基因組測序結果中發現了與噬菌體相似的序列。為了證明原噬菌體的存在，我們使用絲裂黴素C處理NCKUH-21並成功誘導噬菌體的產生。我們將其命名為φNCKUH-21。以前的研究表明，有些噬菌體可以調節艱難梭菌中的毒素基因表現。因此在本研究中，我們想要理解φNCKUH-21在艱難梭菌致病機制中的作用。首先，我們檢測了φNCKUH-21感染艱難梭菌菌株R20291形成溶原體後對毒素基因表現的影響。透過及時定量聚合酶反應進行分析R20291 /φNCKUH-21溶原菌中致病性基因座(PaLoc)主要毒力基因tcdA和tcdB的表達增加至少兩倍。此外，為了識別哪個噬菌體基因（一個或多個）可上調的艱難梭菌毒素表達，三個噬菌體基因NCKUH-21_03888，NCKUH-21_03890，NCKUH-21_03903包括內源性啟動子進行克隆並接合到R20291菌株的毒素基因表現分析。結果表明，由NCKUH-21_03890編碼的CI噬菌體抑制因子可能在毒素基因表現的調節中起作用。我們還選殖了含有6-His的重組蛋白NCKUH-21_03890，通過報告基因測定來研究蛋白質結合位點。總而言之，這些發現表明φNCKUH-21的存在可以影響該醫院重要病原體中的毒素基因調控。

    Abstract
    Clostridium difficile is a gram-positive, anaerobic, spore-forming pathogen, capable of causing severe enteric diseases including antibiotic-associated diarrhea, toxic megacolon and pseudomembranous colitis. In this study we identify a phage-like sequence in the whole genome sequencing results of Taiwan C. difficile ribotype 027 clinical isolate NCKUH-21. To confirm the presence of prophage, NCKUH-21 was treated with mitomycin C and a bacteriophage was induced successfully. We named it as φNCKUH-21. Previous studies have shown that some bacteriophages could modulate the toxin gene production in C. difficile. In this study, we want to comprehend the role of φNCKUH-21 in C. difficile pathogenesis. Therefore, we examined the effect of lysogeny by φNCKUH-21 on C. difficile strain R20291 toxin production. The transcriptional analysis demonstrated an increase at least two-fold in the expression of pathogenicity locus genes tcdA and tcdB in R20291/φNCKUH-21 lysogens. Further, to identify which phage gene(s) could up-regulate the C .difficile toxin expression, three phage genes NCKUH-21_03888, NCKUH-21_03890, NCKUH-21_03903 including the endogenous promoters were cloned and conjugated into R20291 strain for the toxin genes expression assay. The result demonstrated that CI phage repressor which encoded by NCKUH-21_03890 may play a role in the regulation of toxin gene expression. We also cloned a recombinant NCKUH-21_03890 containing 6-His tag to investigate the protein binding site by reporter assay. Together, these findings suggest the presence of φNCKUH-21 can influence toxin gene regulation in this nosocomial important pathogen.

    中文摘要 I Abstract II 誌謝 III Abbreviation IV Figure List IX Table List XI Chapter 1. Introduction 1 Chapter 2. 4 1. Material and Method 4 1.1. Bacterial strains and growth conditions. 4 1.2. Prophage induction 4 1.3. Phage infection 5 1.4. Biofilm formation assay 5 1.5. Bacterial RNA extraction and gene expression analysis 6 1.6. Construction of protein-expressing plasmids in C. difficile and E. coli 7 1.7. Preparation of competent cell (chemical method) 8 1.8. E. coli transformation 9 1.9. C. difficile - E. coli conjugation 9 1.10. C. difficile toxin A and B ELISA 10 1.11. Cell culture 10 1.12. Cytotoxicity assay 11 1.13. Bioluminescence reporter assay 12 1.14. Detection of the gene NCKUH-21_03890 in C. difficile clinical isolated strains 12 1.15. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western Blot 12 1.15.1. Sample preparation 12 1.15.2. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) 13 1.15.3. Western blot 13 1.15.4. Detecting 14 Chapter3 15 2. Results 15 2.1. Prophage induction 15 2.2. Prophage φNCKUH-21 stimulated toxin production and PaLoc gene expression in R20291 lysogen 15 2.3. Identification of phage genes that affect C. difficile PaLoc genes expression 17 2.4. Interaction between NCKUH-21_03890 or NCKUH-21_03903 protein with tcdR promoter 18 2.5. Induction and purification of recombinant protein 20 2.6. Glucose does not have the ability to promote NCKUH-21_03890 protein production. 21 2.7. Biofilm formation of R20291 lysogen and R20291 21 2.8. Prevalence of NCKUH-21_03890 in clinical C. difficile strains in Taiwan. 22 Chapter 4 23 3. Discussion 23 3.1. Prophage modulate the toxin production and PaLoc gene expression 23 Reference 29 Figure 33 Table 52 Supplementary Materials 59 Compounds used in this study 64 Powder used in this study 65

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