簡易檢索 / 詳目顯示

回結果列表
研究生： 陳冠宇
Chen, Kuan-Yu
論文名稱： 探討在困難梭狀芽孢桿菌中調控因子白胺酸反應調控蛋白之功能
Characterization of an Lrp/ AsnC family regulator in Clostridium difficile
指導教授： 黃一修
Huang, I-Hsiu
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： 2017
畢業學年度： 105
語文別： 英文
論文頁數： 63
中文關鍵詞： 艱難梭狀芽孢桿菌 、白胺酸調控蛋白 、毒素A/毒素B 、孢子生成 、運動 、突變系統
外文關鍵詞： Clostridium difficile, toxin, Leucine-responsive regulatory protein, sporulation, motility, genetic system
相關次數： 點閱：98  下載：1 
分享至:
查詢本校圖書館目錄 查詢臺灣博碩士論文知識加值系統 勘誤回報

    • 艱難梭狀芽孢桿菌屬於革蘭氏陽性菌、會產生孢子的細菌，且在院內造成腹瀉的主要原因。目前已經許多的致病因子都有文獻指出與艱難梭狀桿菌的感染有關，像是毒素、孢子形成、鞭毛以及細菌的第四型線毛。在過去，已經有許多轉錄因子被研究。然而，在艱難梭狀芽孢桿菌得基因組中存在許多轉錄調控因子並且都涉及於毒素的產生。在許多革蘭氏陰性菌中已知白胺酸調控蛋白參與調節許多基因表達。在研究的第一部分中從艱難梭狀桿菌的基因體中也鑑定到具有白胺酸調控蛋白，其扮演在毒力的角色是未知的。將此基因突變發現到此蛋白在細菌生長時對於養分是必要的。此蛋白也被證明會抑制毒素A和毒素B的產生。細胞毒性測定以及小鼠實驗中也發現到白胺酸調控蛋白參與艱難梭菌的發病機制。有趣的是當白胺酸蛋白缺失時，細菌的游動與孢子化皆會受到影響，但此現象只存在於菌屬之間。在研究的第二部分中，我們優化艱難梭狀桿菌的遺傳系統，成功建構無標記之質體以及雙突變株。總結以上，我們首次證明艱難梭菌中的白胺酸調控蛋白是一個新型的調控子，參與在細胞生長、毒素生成、孢子形成和運動性之中。此外我們也建構出一個新穎的突變系統。

    Clostridium difficile is a gram positive, spore-forming bacterium and the major cause of nosocomial diarrhea. Multiple virulence factors including toxins, spore formation, flagella, Type IV pili, and many others have been reported to contribute to the pathogenesis of C. difficile infection. In the past, several transcriptional regulators of these virulence factors have been identified. However, a plethora of genes encoding for transcriptional regulators are present in the genome of C. difficile and it is likely that many are also involved in virulence regulation. The Leucine responsive regulatory protein (Lrp) is known to regulate a wide variety of genes in numerous gram-negative bacteria. In part 1 of this study, a Lrp homologue was identified in the genome of C. difficile but its role in virulence was unknown. Mutational analysis revealed that Lrp is required for growth under nutrient limiting condition. Importantly, Lrp was also shown to be a repressor of both toxin A and toxin B production. Murine model of infection and cytotoxicity assay demonstrated that Lrp is involved in the pathogenesis of C. difficile in vivo and in vitro. Interestingly, swimming motility and sporulation were also affected by the inactivation of Lrp but the effect appeared to be strain specific. In part 2 of this study, we thought to optimize the current genetic system available in C. difficile by creating a markerless mutator plasmid and succeed in generating double mutants without any marker selection. In summary, we have demonstrated for the first time that the Lrp in C. difficile is a novel regulator of cell growth, toxin production, sporulation, and motility. In addition, we have also generated a novel tool for creating multiple mutants in C. difficile.

    Abstract I 中文摘要 II 誌謝 III Abbreviations IX Chapter 1 1 Introduction 1 1.1 Clostridium difficile infection 1 1.2 Diagnosis and treatment of CDI 1 1.3 C. difficile pathogenesis 2 1.4 Known regulators of C. difficile toxins 3 1.5 Leucine-Responsive Regulatory Protein (Lrp) 3 1.6 Rationale 4 Chapter 2 5 Materials and Methods 5 2.1 Bacterial strains, plasmid and growth conditions 5 2.2 Polymerase chain reaction (PCR) primers 5 2.3 High fidelity PCR 5 2.4 Agarose gel electrophoresis 6 2.5 Restriction enzyme digestion 6 2.6 DNA ligation 6 2.7 Heat-shock transformation of E. coli 7 2.8 Extraction of genomic DNA from C. difficile 7 2.9 E. coli – C. difficile conjugation 8 2.10 ClosTron mutagenesis of lrp 8 2.11 PCR screening for ligation 9 2.12 Growth curve 9 2.13 RNA extraction and qRT-PCR 10 2.14 Western blot analysis 11 2.15 Mouse model of C. difficile infection 12 2.16 Motility assay 13 2.17 Cell culture 13 2.18 Sporulation assay 14 2.19 Markerless ClosTron genetic system construction 14 Chapter 3 16 Part I: Characterization of Lrp in C. difficile 16 Result 16 3.1 Identification of Lrp/ AsnC family regulators in C. difficile 16 3.2 Construction of lrp insertional mutant and complementation strains 17 3.3 The role of Lrp in C. difficile growth 18 3.4 Lrp repressed C. difficile toxin gene expression 18 3.5 lrp mutant is more cytotoxic to Vero and Caco-2 cells than wild type in both strain R20291 and DPS630 18 3.6 Lrp is involved in C. difficile pathogenesis in vivo 19 3.7 lrp is involved in C. difficile motility 20 3.8 The role of Lrp in C. difficile sporulation 20 Chapter 3 21 Part 2: Optimizing genetic system in C. difficile 21 Creation of a markerless genetic system 21 Chapter 4 23 Discussion 23 References 28 Tables. 33 Figures. 44 Supplementary Materials 61

    1. Rupnik M, Wilcox MH, & Gerding DN (2009) Clostridium difficile infection: new developments in epidemiology and pathogenesis. Nat Rev Microbiol 7(7):526-536.
    2. Lo Vecchio A & Zacur GM (2012) Clostridium difficile infection: an update on epidemiology, risk factors, and therapeutic options. Curr Opin Gastroenterol 28(1):1-9.
    3. Kelly CP & LaMont JT (2008) Clostridium difficile--more difficult than ever. N Engl J Med 359(18):1932-1940.
    4. Ofosu A (2016) Clostridium difficile infection: a review of current and emerging therapies. Ann Gastroenterol 29(2):147-154.
    5. George WL, Sutter VL, Citron D, & Finegold SM (1979) Selective and differential medium for isolation of Clostridium difficile. J Clin Microbiol 9(2):214-219.
    6. Delmee M (2001) Laboratory diagnosis of Clostridium difficile disease. Clin Microbiol Infect 7(8):411-416.
    7. Bartlett JG & Gerding DN (2008) Clinical recognition and diagnosis of Clostridium difficile infection. Clin Infect Dis 46 Suppl 1:S12-18.
    8. Cohen SH, et al. (2010) Clinical practice guidelines for Clostridium difficile infection in adults: 2010 update by the society for healthcare epidemiology of America (SHEA) and the infectious diseases society of America (IDSA). Infect Control Hosp Epidemiol 31(5):431-455.
    9. Jarrad AM, Karoli T, Blaskovich MA, Lyras D, & Cooper MA (2015) Clostridium difficile drug pipeline: challenges in discovery and development of new agents. J Med Chem 58(13):5164-5185.
    10. Rubio-Terres C, et al. (2015) Economic assessment of fidaxomicin for the treatment of Clostridium difficile infection (CDI) in special populations (patients with cancer, concomitant antibiotic treatment or renal impairment) in Spain. Eur J Clin Microbiol Infect Dis 34(11):2213-2223.
    11. Khoruts A, Dicksved J, Jansson JK, & Sadowsky MJ (2010) Changes in the composition of the human fecal microbiome after bacteriotherapy for recurrent Clostridium difficile-associated diarrhea. J Clin Gastroenterol 44(5):354-360.
    12. Kelly CR, et al. (2016) Effect of Fecal Microbiota Transplantation on Recurrence in Multiply Recurrent Clostridium difficile Infection: A Randomized Trial. Ann Intern Med 165(9):609-616.
    13. Edwards AN, et al. (2016) Chemical and Stress Resistances of Clostridium difficile Spores and Vegetative Cells. Front Microbiol 7:1698.
    14. Braun V, Hundsberger T, Leukel P, Sauerborn M, & von Eichel-Streiber C (1996) Definition of the single integration site of the pathogenicity locus in Clostridium difficile. Gene 181(1-2):29-38.
    15. Chen S, Sun C, Wang H, & Wang J (2015) The Role of Rho GTPases in Toxicity of Clostridium difficile Toxins. Toxins (Basel) 7(12):5254-5267.
    16. Yang Z, Zhang Y, Huang T, & Feng H (2015) Glucosyltransferase activity of Clostridium difficile Toxin B is essential for disease pathogenesis. Gut Microbes 6(4):221-224.
    17. Voth DE & Ballard JD (2005) Clostridium difficile toxins: mechanism of action and role in disease. Clin Microbiol Rev 18(2):247-263.
    18. Bouillaut L, Dubois T, Sonenshein AL, & Dupuy B (2015) Integration of metabolism and virulence in Clostridium difficile. Res Microbiol 166(4):375-383.
    19. Mani N & Dupuy B (2001) Regulation of toxin synthesis in Clostridium difficile by an alternative RNA polymerase sigma factor. Proc Natl Acad Sci U S A 98(10):5844-5849.
    20. Matamouros S, England P, & Dupuy B (2007) Clostridium difficile toxin expression is inhibited by the novel regulator TcdC. Mol Microbiol 64(5):1274-1288.
    21. Antunes A, Martin-Verstraete I, & Dupuy B (2011) CcpA-mediated repression of Clostridium difficile toxin gene expression. Mol Microbiol 79(4):882-899.
    22. Dineen SS, Villapakkam AC, Nordman JT, & Sonenshein AL (2007) Repression of Clostridium difficile toxin gene expression by CodY. Mol Microbiol 66(1):206-219.
    23. Mackin KE, Carter GP, Howarth P, Rood JI, & Lyras D (2013) Spo0A differentially regulates toxin production in evolutionarily diverse strains of Clostridium difficile. PLoS One 8(11):e79666.
    24. Purcell EB, McKee RW, McBride SM, Waters CM, & Tamayo R (2012) Cyclic diguanylate inversely regulates motility and aggregation in Clostridium difficile. J Bacteriol 194(13):3307-3316.
    25. Wang Q, Wu J, Friedberg D, Plakto J, & Calvo JM (1994) Regulation of the Escherichia coli lrp gene. J Bacteriol 176(7):1831-1839.
    26. Ferrario M, et al. (1995) The leucine-responsive regulatory protein of Escherichia coli negatively regulates transcription of ompC and micF and positively regulates translation of ompF. J Bacteriol 177(1):103-113.
    27. Shimada T, Saito N, Maeda M, Tanaka K, & Ishihama A (2015) Expanded roles of leucine-responsive regulatory protein in transcription regulation of the Escherichia coli genome: Genomic SELEX screening of the regulation targets. Microbial Genomics 1(1).
    28. Beloin C, et al. (2000) Characterization of LrpC DNA-binding properties and regulation of Bacillus subtilis lrpC gene expression. J Bacteriol 182(16):4414-4424.
    29. Beloin C, et al. (1997) Characterization of an lrp-like (lrpC) gene from Bacillus subtilis. Mol Gen Genet 256(1):63-71.
    30. Tapias A, Lopez G, & Ayora S (2000) Bacillus subtilis LrpC is a sequence-independent DNA-binding and DNA-bending protein which bridges DNA. Nucleic Acids Res 28(2):552-559.
    31. Baek CH, Wang S, Roland KL, & Curtiss R, 3rd (2009) Leucine-responsive regulatory protein (Lrp) acts as a virulence repressor in Salmonella enterica serovar Typhimurium. J Bacteriol 191(4):1278-1292.
    32. Reddy MC, Gokulan K, Jacobs WR, Jr., Ioerger TR, & Sacchettini JC (2008) Crystal structure of Mycobacterium tuberculosis LrpA, a leucine-responsive global regulator associated with starvation response. Protein Sci 17(1):159-170.
    33. Hay NA, Tipper DJ, Gygi D, & Hughes C (1997) A nonswarming mutant of Proteus mirabilis lacks the Lrp global transcriptional regulator. J Bacteriol 179(15):4741-4746.
    34. Williams DR, Young DI, & Young M (1990) Conjugative plasmid transfer from Escherichia coli to Clostridium acetobutylicum. J Gen Microbiol 136(5):819-826.
    35. Heap JT, Pennington OJ, Cartman ST, Carter GP, & Minton NP (2007) The ClosTron: a universal gene knock-out system for the genus Clostridium. J Microbiol Methods 70(3):452-464.
    36. Kuehne SA & Minton NP (2012) ClosTron-mediated engineering of Clostridium. Bioengineered 3(4):247-254.
    37. Lawley TD, et al. (2009) Antibiotic treatment of Clostridium difficile carrier mice triggers a supershedder state, spore-mediated transmission, and severe disease in immunocompromised hosts. Infect Immun 77(9):3661-3669.
    38. Ng YK, et al. (2013) Expanding the repertoire of gene tools for precise manipulation of the Clostridium difficile genome: allelic exchange using pyrE alleles. PLoS One 8(2):e56051.
    39. Heap JT, Pennington OJ, Cartman ST, & Minton NP (2009) A modular system for Clostridium shuttle plasmids. J Microbiol Methods 78(1):79-85.
    40. 邱意晴 & Chiu Y-C (白胺酸反應調控蛋白為困難梭狀芽孢桿菌之毒力廣泛調控子.).

    下載圖示 校內：2019-08-31公開
    校外：2019-08-31公開
    QR CODE