簡易檢索 / 詳目顯示

回結果列表
研究生： 周文怡
論文名稱： 創傷弧菌高溶血突變株之鑑定與反應調節基因表現分析
Characterization of a hyperhemolytic mutant and expression analysis of response regulator genes in Vibrio vulnificus
指導教授： 張晃猷 博士
口試委員:
學位類別： 碩士
Master
系所名稱： 生命科學暨醫學院 - 分子醫學研究所
Institute of Molecular Medicine
論文出版年： 2004
畢業學年度： 92
語文別： 中文
論文頁數： 50
中文關鍵詞： 創傷弧菌 、轉位子標定突變法 、反應調節基因
相關次數： 點閱：2  下載：0 
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

    • 中文摘要
    創傷弧菌是一種存在於海水中的革蘭氏陰性菌，在免疫缺損的病患能造成高死亡率。此菌含多種與致病有關的基因，為了了解其致病原理，最基本的工作就是先找出創傷弧菌的基因體中有哪些基因參與致病過程，以及致病過程中所扮演的角色。我們建立了一套適用於創傷弧菌的轉位子標定突變法，以卡那黴素抗藥性作為篩選的指標，構築一個轉位子突變株庫。將此突變株庫利用不同的生長環境篩選，得到一個具有高溶血能力的突變株WY005。為了了解是哪個基因受到轉位子插入而造成表型的改變，我將此菌的染色體DNA經部分接割後，粘接至載體，獲得轉位子所插入的基因片段。由序列分析得知，此基因為創傷弧菌質體pYJ016的pilT。PilT蛋白質是形成第四型纖毛的組成分之一，協助細菌運動及附著，文獻指出pilT突變株與細胞毒性有關，可能因此使得WY005具有破壞紅血球的能力 。此外，我選殖了創傷弧菌中的38個反應調節基因(response regulator)到pET表現載體上，並成功的表現出28種蛋白質；同時利用RNA dot blot的方法分析一些反應調節基因在不同生長條件下的表現量。結果發現phoB及gltR基因表現量在缺鐵及營養缺乏的條件下明顯的增加。此結果顯示此兩基因表現可能與創傷弧菌抵抗營養缺乏環境有關。

    Abstract
    Vibrio vulnificus, a gram-negative marine bacterium, is an opportunistic pathogen with a high invasive capability that causes high mortality in infected immunocompromised patients. In order to solve the problem of the bacterial infection, it is essential to understand the pathogenesis of the pathogen, particularly the virulence-associated genes and their roles in the infection process. In this study we have established a kanamycin resistance gene-based transposon mutagenesis system for use in V. vulnificus. A mutant WY005 which exhibited a high hemolytic ability was isolated. The transposon insertion site was cloned by seletion on kanamycin containing agar and the identity of the disrupted gene was determined by nucleotide sequencing. Sequence comparison in the GenBank database has demonstrated that the gene is pilT that is located on V. vulnificus plasmid pYJ016. PilT is a putative nucleotide-binding protein involved in function of type IV pili including movement and attachment to the host. In Neisseria, a PilT mutant has been reported to participate in release of heme from red blood cells. We also attempt to investigate the expression of response regulator gene in V.vulnificus. A total of 38 response regulators of V. vulnificus that contain a putative DNA binding motif were cloned and expressed. Among them 28 has successfully yielded a protein of expected sizes. Finally, using the RNA dot blotting, expression levels of 7 response regulator genes under different growth conditions were analyzed. The expression level of PhoB and gltR genes were found to be activated significantly under ferric limitation and minimum medium culture. The results suggest that the two response regulators play a role in counter nutrient limitation conditions in V.vulnificus.

    Contents Abstract (Chinese) i Abstract (English) ii Contents iv Table contents vii Figure contents viii Appendix content x Abbreviation xi Chapter 1. Introduction 1 1. Vibrio vulnificus 1 2. Biotypes 1 3. Host susceptibility 2 4. Clinical symptoms and treatment 3 5. virulence of V. vulnificus 4 6 Two component system 7 7 This study 9 Chapter 2. Materials and Methods 12 Bacterial strains and plasmids 12 Media, cultivation and storage of bacteria 12 Construction of Tn10 V. vulnificus YJ016 mutant libiary 12 Microbial sensitivity assays 12 Genomic DNA preparation and PCR 13 Southern hybridization 13 Bioinformatics analysis 14 Construction of expression plasmids of the response regulator 14 Expression of recombinant protein in E. coli 15 RNA preparation 15 RNA blotting 16 Preparation of 32P labeled probes 17 Hybridization of arrays and image analysis 17 Chapter 3. Results 19 Part 1 Isolation of Tn10 mutants of V. vulnificus 19 WY005 mutant has high hemolysis ability 19 WY005 mutant has a faster growth rate than the wild type 20 WY005 has a higher hemolytic activity under low-salt growth condition 20 Susceptiblity of V. vulnificus YJ016 and WY005 to antimicrobial agents 21 Southern blotting analysis and identification of the Tn10 insertion site in WY005 21 LF005 mutant doesn’t shown a high hemolytic activity 22 Part 2 Overexpression of 38 response regulators in E. coli 22 Part 3 Relative expression levels of the response regulators at different stress treatments 23 Chapter 4. Discussion 25 References 30 Tables and Figures 34 Appendix 50 Table contents Table1. Bacterial strains and plasmids used in this study…...34 Table2. Primers used in this study………………….35 Table3. The response regulator genes investigated in this study…………………………………………………………...36 Table4. Sensitivity of YJ016 and WY005 to antimicrobial agents………………………………………………………….37 Figure contents FIG.1. Hemolytic activity of WY005 and YJ016 on blood agar plate……………………………………….…………………..38 FIG.2. Growth curves of V. vulnificus YJ016 and WY005….39 FIG.3. Lysis of RBC’s by bacteria grown under different conditions……………………………….…………………….40 FIG.4. Sensitivity of V. vulnificus YJ016 and WY005 to antimicrobial agents…………………………………………..41 FIG.5. Southern blot analysis of the WY005 with differentrestrictionenzymes…………………………………...42 FIG.6. Schematic map of Tn10 and the flanking region…….43 FIG.7. Neucleotide sequence alignment of WY007 and pilT of Vibrio vulnificus YJ016……………………………………….44 FIG.8. Hemolytic activity of LF005 and YJ016 on blood agar plate……………………………………………………………45 FIG.9. 38 response regulator gene products amplification by PCR………………………………………………….….46 FIG.10. Expression of V. vulnificus response regulator genes in E. coli………………………………………………………....47 FIG.11. RNA dot blot ………………………………………48 FIG.12. Quantification of phoB and gilT gene expression….49 Appendix content Appendix 1. The pET101/D-TOPO expression vector……50

    REFRENCES
    1. Amaro, C., and E. G. Biosca. 1996. Vibrio vulnificus biotype 2, pathogenic for eels, is also an opportunistic pathogen for humans. Appl Environ Microbiol 62:1454-7.
    2. Amaro, C., E. G. Biosca, B. Fouz, A. E. Toranzo, and E. Garay. 1994. Role of iron, capsule, and toxins in the pathogenicity of Vibrio vulnificus biotype 2 for mice. Infect Immun 62:759-63.
    3. Belitsky, B. R., and A. L. Sonenshein. 1997. Altered transcription activation specificity of a mutant form of Bacillus subtilis GltR, a LysR family member. J Bacteriol 179:1035-43.
    4. Chen, C. J., D. M. Tobiason, C. E. Thomas, W. M. Shafer, H. S. Seifert, and P. F. Sparling. 2004. A mutant form of the Neisseria gonorrhoeae pilus secretin protein PilQ allows increased entry of heme and antimicrobial compounds. J Bacteriol 186:730-9.
    5. Cornelissen, C. N., and P. F. Sparling. 1994. Iron piracy: acquisition of transferrin-bound iron by bacterial pathogens. Mol Microbiol 14:843-50.
    6. Gray, L. D., and A. S. Kreger. 1985. Purification and characterization of an extracellular cytolysin produced by Vibrio vulnificus. Infect Immun 48:62-72.
    7. Helms, S. D., J. D. Oliver, and J. C. Travis. 1984. Role of heme compounds and haptoglobin in Vibrio vulnificus pathogenicity. Infect Immun 45:345-9.
    8. Henikoff, S., G. W. Haughn, J. M. Calvo, and J. C. Wallace. 1988. A large family of bacterial activator proteins. Proc Natl Acad Sci U S A 85:6602-6.
    9. Klontz, K. C., S. Lieb, M. Schreiber, H. T. Janowski, L. M. Baldy, and R. A. Gunn. 1988. Syndromes of Vibrio vulnificus infections. Clinical and epidemiologic features in Florida cases, 1981-1987. Ann Intern Med 109:318-23.
    10. Koenig, K. L., J. Mueller, and T. Rose. 1991. Vibrio vulnificus. Hazard on the half shell. West J Med 155:400-3.
    11. Kothary, M. H., and A. S. Kreger. 1987. Purification and characterization of an elastolytic protease of Vibrio vulnificus. J Gen Microbiol 133 ( Pt 7):1783-91.
    12. Kumar, A., B. Grimes, N. Fujita, K. Makino, R. A. Malloch, R. S. Hayward, and A. Ishihama. 1994. Role of the sigma 70 subunit of Escherichia coli RNA polymerase in transcription activation. J Mol Biol 235:405-13.
    13. Maier, B., L. Potter, M. So, C. D. Long, H. S. Seifert, and M. P. Sheetz. 2002. Single pilus motor forces exceed 100 pN. Proc Natl Acad Sci U S A 99:16012-7.
    14. Makino, K., H. Shinagawa, M. Amemura, T. Kawamoto, M. Yamada, and A. Nakata. 1989. Signal transduction in the phosphate regulon of Escherichia coli involves phosphotransfer between PhoR and PhoB proteins. J Mol Biol 210:551-9.
    15. Makino, K., H. Shinagawa, M. Amemura, and A. Nakata. 1986. Nucleotide sequence of the phoR gene, a regulatory gene for the phosphate regulon of Escherichia coli. J Mol Biol 192:549-56.
    16. Makino, K., H. Shinagawa, and A. Nakata. 1985. Regulation of the phosphate regulon of Escherichia coli K-12: regulation and role of the regulatory gene phoR. J Mol Biol 184:231-40.
    17. Merz, A. J., M. So, and M. P. Sheetz. 2000. Pilus retraction powers bacterial twitching motility. Nature 407:98-102.
    18. Miyoshi, N., C. Shimizu, S. Miyoshi, and S. Shinoda. 1987. Purification and characterization of Vibrio vulnificus protease. Microbiol Immunol 31:13-25.
    19. Moreno, M. L., and M. Landgraf. 1998. Virulence factors and pathogenicity of Vibrio vulnificus strains isolated from seafood. J Appl Microbiol 84:747-51.
    20. Parkinson, J. S., and E. C. Kofoid. 1992. Communication modules in bacterial signaling proteins. Annu Rev Genet 26:71-112.
    21. Perego, M., C. Hanstein, K. M. Welsh, T. Djavakhishvili, P. Glaser, and J. A. Hoch. 1994. Multiple protein-aspartate phosphatases provide a mechanism for the integration of diverse signals in the control of development in B. subtilis. Cell 79:1047-55.
    22. Perego, M., and J. A. Hoch. 1996. Cell-cell communication regulates the effects of protein aspartate phosphatases on the phosphorelay controlling development in Bacillus subtilis. Proc Natl Acad Sci U S A 93:1549-53.
    23. Perego, M., and J. A. Hoch. 1996. Protein aspartate phosphatases control the output of two-component signal transduction systems. Trends Genet 12:97-101.
    24. Powell, J. L., A. C. Wright, S. S. Wasserman, D. M. Hone, and J. G. Morris, Jr. 1997. Release of tumor necrosis factor alpha in response to Vibrio vulnificus capsular polysaccharide in in vivo and in vitro models. Infect Immun 65:3713-8.
    25. Rodrigues, D. P., R. V. Ribeiro, and E. Hofer. 1992. Analysis of some virulence factors of Vibrio vulnificus isolated from Rio de Janeiro, Brazil. Epidemiol Infect 108:463-7.
    26. Russel, M. 1998. Macromolecular assembly and secretion across the bacterial cell envelope: type II protein secretion systems. J Mol Biol 279:485-99.
    27. Sanders, D. A., B. L. Gillece-Castro, A. L. Burlingame, and D. E. Koshland, Jr. 1992. Phosphorylation site of NtrC, a protein phosphatase whose covalent intermediate activates transcription. J Bacteriol 174:5117-22.
    28. Schell, M. A. 1993. Molecular biology of the LysR family of transcriptional regulators. Annu Rev Microbiol 47:597-626.
    29. Schreier, H. J. 1993. Biosynthesis of glutamine and glutamate and assimilation of ammonia. In J. A. H. In A. L. Sonenshein, and R. Losick (ed.) (ed.), Bacillus subtilis and other gram-positive bacteria: biochemistry, physiology, and molecular genetics. American Society for Microbiology, Washington, D.C.
    30. Schryvers, A. B., and I. Stojiljkovic. 1999. Iron acquisition systems in the pathogenic Neisseria. Mol Microbiol 32:1117-23.
    31. Shapiro, R. L., S. Altekruse, L. Hutwagner, R. Bishop, R. Hammond, S. Wilson, B. Ray, S. Thompson, R. V. Tauxe, and P. M. Griffin. 1998. The role of Gulf Coast oysters harvested in warmer months in Vibrio vulnificus infections in the United States, 1988-1996. Vibrio Working Group. J Infect Dis 178:752-9.
    32. Shimizu, T., W. Ba-Thein, M. Tamaki, and H. Hayashi. 1994. The virR gene, a member of a class of two-component response regulators, regulates the production of perfringolysin O, collagenase, and hemagglutinin in Clostridium perfringens. J Bacteriol 176:1616-23.
    33. Shinagawa, H., K. Makino, M. Amemura, and A. Nakata. 1987. Structure and function of the regulatory genes for the phosphate regulon in Escherichia coli. In F. G. R. In A. Torriani-Gorini, S. Silver, A. Wright, and and E. Yagil, Phosphate metabolism and cellular regulation in microorganisms. American Society for Microbiology, Washington, D.C.
    34. Simpson, L. M., V. K. White, S. F. Zane, and J. D. Oliver. 1987. Correlation between virulence and colony morphology in Vibrio vulnificus. Infect Immun 55:269-72.
    35. Smith, G. C., and J. R. Merkel. 1982. Collagenolytic activity of Vibrio vulnificus: potential contribution to its invasiveness. Infect Immun 35:1155-6.
    36. Stelma, G. N., Jr., A. L. Reyes, J. T. Peeler, C. H. Johnson, and P. L. Spaulding. 1992. Virulence characteristics of clinical and environmental isolates of Vibrio vulnificus. Appl Environ Microbiol 58:2776-82.
    37. Tison, D. L., M. Nishibuchi, J. D. Greenwood, and R. J. Seidler. 1982. Vibrio vulnificus biogroup 2: new biogroup pathogenic for eels. Appl Environ Microbiol 44:640-6.
    38. Veenstra, J., P. J. Rietra, J. Goudswaard, J. A. Kaan, P. H. van Keulen, and C. P. Stoutenbeek. 1993. [Extra-intestinal infections caused by Vibrio spp. in The Netherlands]. Ned Tijdschr Geneeskd 137:654-7.
    39. Wanner, B. L. 1996. Phosphorous assimilation and control of the phosphate regulon. In R. C. I. In F. C. Neidhardt, J. L. Ingraham, E. C. C. Lin, K. B. Low, B. Magasanik, W. S. Reznikoff, M. Riley, M. Schaechter, and H. E. Umbarger, Escherichia coli and Salmonella: cellular and molecular biology. American Society for Microbiology, Washington, D.C.
    40. Weinberg, E. D. 1978. Iron and infection. Microbiol Rev 42:45-66.
    41. Wright, A. C., L. M. Simpson, and J. D. Oliver. 1981. Role of iron in the pathogenesis of Vibrio vulnificus infections. Infect Immun 34:503-7.
    42. Wright, A. C., L. M. Simpson, J. D. Oliver, and J. G. Morris, Jr. 1990. Phenotypic evaluation of acapsular transposon mutants of Vibrio vulnificus. Infect Immun 58:1769-73.
    43. Yoshida, S., M. Ogawa, and Y. Mizuguchi. 1985. Relation of capsular materials and colony opacity to virulence of Vibrio vulnificus. Infect Immun 47:446-51.

    無法下載圖示 全文公開日期 本全文未授權公開 (校內網路)
    全文公開日期 本全文未授權公開 (校外網路)

    QR CODE