請用此 Handle URI 來引用此文件：
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/54069完整後設資料紀錄
| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 王錦堂(Jin-Town Wang) | |
| dc.contributor.author | Pei-Yin Liu | en |
| dc.contributor.author | 劉沛吟 | zh_TW |
| dc.date.accessioned | 2021-06-16T02:38:45Z | - |
| dc.date.available | 2020-09-25 | |
| dc.date.copyright | 2015-09-25 | |
| dc.date.issued | 2015 | |
| dc.date.submitted | 2015-07-23 | |
| dc.identifier.citation | 1. Murray PR, Rosenthal KS, Kobayashi GS and Pfaller MA. Enterobacteriaceae. Medical Microbiology. 3rd ed: Mosby, 1998:232-244. 2. Pan, Y.J., et al., Capsular polysaccharide synthesis regions in Klebsiella pneumoniae serotype K57 and a new capsular serotype. J Clin Microbiol, 2008. 46(7): p. 2231-40. 3. Edmondson, A.S. and E.M. Cooke, The development and assessment of a bacteriocin typing method for Klebsiella. J Hyg (Lond), 1979. 82(2): p. 207-23. 4. Hsu, C.R., et al., Isolation of a bacteriophage specific for a new capsular type of Klebsiella pneumoniae and characterization of its polysaccharide depolymerase. PLoS One, 2013. 8(8): p. e70092. 5. Fung, C.P., et al., A 5-year study of the seroepidemiology of Klebsiella pneumoniae: high prevalence of capsular serotype K1 in Taiwan and implication for vaccine efficacy. J Infect Dis, 2000. 181(6): p. 2075-9. 6. Bergogne-Berezin, E., Treatment and prevention of nosocomial pneumonia. Chest, 1995. 108(2 Suppl): p. 26S-34S. 7. Kaushik, S.L., et al., Neonatal sepsis in hospital born babies. J Commun Dis, 1998. 30(3): p. 147-52. 8. Yinnon, A.M., et al., Klebsiella bacteraemia: community versus nosocomial infection. QJM, 1996. 89(12): p. 933-41. 9. Fang, C.T., et al., A novel virulence gene in Klebsiella pneumoniae strains causing primary liver abscess and septic metastatic complications. J Exp Med, 2004. 199(5): p. 697-705. 10. Carpenter, J. L. Klebsiella pulmonary infections: occurrence at one medical center and review. Rev Infect Dis, 1990.12:672-82. 11. Cheng, D.L., et al., Pyogenic liver abscess: clinical manifestations and value of percutaneous catheter drainage treatment. J Formos Med Assoc, 1990. 89(7): p. 571-6. 12. Wang, J.H., et al., Primary liver abscess due to Klebsiella pneumoniae in Taiwan. Clin Infect Dis, 1998. 26(6): p. 1434-8. 13. Fung, C.P., et al., A global emerging disease of Klebsiella pneumoniae liver abscess: is serotype K1 an important factor for complicated endophthalmitis? Gut, 2002. 50(3): p. 420-4. 14. Mizuta, K., et al., Virulence for mice of Klebsiella strains belonging to the O1 group: relationship to their capsular (K) types. Infect Immun, 1983. 40(1): p. 56-61. 15. Domenico, P., et al., Polysaccharide capsule-mediated resistance to opsonophagocytosis in Klebsiella pneumoniae. Infect Immun, 1994. 62(10): p. 4495-9. 16. Merino, S., et al., Mechanisms of Klebsiella pneumoniae resistance to complement-mediated killing. Infect Immun, 1992. 60(6): p. 2529-35. 17. Podschun, R. and U. Ullmann, Klebsiella spp. as nosocomial pathogens: epidemiology, taxonomy, typing methods, and pathogenicity factors. Clin Microbiol Rev, 1998. 11(4): p. 589-603. 18. Alvarez, D., et al., Capsular polysaccharide is a major complement resistance factor in lipopolysaccharide O side chain-deficient Klebsiella pneumoniae clinical isolates. Infect Immun, 2000. 68(2): p. 953-5. 19. Tomas, J.M., et al., Role of capsule and O antigen in resistance of Klebsiella pneumoniae to serum bactericidal activity. Infect Immun, 1986. 54(1): p. 85-9. 20. McCallum, K.L., et al., A high-molecular-weight fraction of smooth lipopolysaccharide in Klebsiella serotype O1:K20 contains a unique O-antigen epitope and determines resistance to nonspecific serum killing. Infect Immun, 1989. 57(12): p. 3816-22. 21. Straus, D.C., D.L. Atkisson, and C.W. Garner, Importance of a lipopolysaccharide-containing extracellular toxic complex in infections produced by Klebsiella pneumoniae. Infect Immun, 1985. 50(3): p. 787-95. 22. Maayan, M.C., et al., Population shift in mannose-specific fimbriated phase of Klebsiella pneumoniae during experimental urinary tract infection in mice. Infect Immun, 1985. 49(3): p. 785-9. 23. Fader, R.C., et al., Evidence that in vitro adherence of Klebsiella pneumoniae to ciliated hamster tracheal cells is mediated by type 1 fimbriae. Infect Immun, 1988. 56(11): p. 3011-3. 24. Sebghati, T.A., et al., Characterization of the type 3 fimbrial adhesins of Klebsiella strains. Infect Immun, 1998. 66(6): p. 2887-94. 25. Gerlach, G.F., S. Clegg, and B.L. Allen, Identification and characterization of the genes encoding the type 3 and type 1 fimbrial adhesins of Klebsiella pneumoniae. J Bacteriol, 1989. 171(3): p. 1262-70. 26. Darfeuille-Michaud, A., et al., R-plasmid-encoded adhesive factor in Klebsiella pneumoniae strains responsible for human nosocomial infections. Infect Immun, 1992. 60(1): p. 44-55. 27. Di Martino, P., et al., Molecular characterization and adhesive properties of CF29K, an adhesin of Klebsiella pneumoniae strains involved in nosocomial infections. Infect Immun, 1995. 63(11): p. 4336-44. 28. Di Martino, P., et al., A new fimbrial antigen harbored by CAZ-5/SHV-4-producing Klebsiella pneumoniae strains involved in nosocomial infections. Infect Immun, 1996. 64(6): p. 2266-73. 29. Perry, R.D. and C.L. San Clemente, Siderophore synthesis in Klebsiella pneumoniae and Shigella sonnei during iron deficiency. J Bacteriol, 1979. 140(3): p. 1129-32. 30. Chou, H.C., et al., Isolation of a chromosomal region of Klebsiella pneumoniae associated with allantoin metabolism and liver infection. Infect Immun, 2004. 72(7): p. 3783-92. 31. Ma, L.C., et al., Genomic heterogeneity in Klebsiella pneumoniae strains is associated with primary pyogenic liver abscess and metastatic infection. J Infect Dis, 2005. 192(1): p. 117-28. 32. Hsieh, P.F., et al., Serum-induced iron-acquisition systems and TonB contribute to virulence in Klebsiella pneumoniae causing primary pyogenic liver abscess. J Infect Dis, 2008. 197(12): p. 1717-27. 33. Lai, Y.C., H.L. Peng, and H.Y. Chang, RmpA2, an activator of capsule biosynthesis in Klebsiella pneumoniae CG43, regulates K2 cps gene expression at the transcriptional level. J Bacteriol, 2003. 185(3): p. 788-800. 34. Hsieh, P.F., et al., Lipopolysaccharide O1 antigen contributes to the virulence in Klebsiella pneumoniae causing pyogenic liver abscess. PLoS One, 2012. 7(3): p. e33155. 35. Niemann, H.H., W.D. Schubert, and D.W. Heinz, Adhesins and invasins of pathogenic bacteria: a structural view. Microbes Infect, 2004. 6(1): p. 101-12. 36. Pizarro-Cerda, J. and P. Cossart, Bacterial adhesion and entry into host cells. Cell, 2006. 124(4): p. 715-27. 37. Gerlach, R.G. and M. Hensel, Protein secretion systems and adhesins: the molecular armory of Gram-negative pathogens. Int J Med Microbiol, 2007. 297(6): p. 401-15. 38. Oelschlaeger, T.A., Adhesins as invasins. Int J Med Microbiol, 2001. 291(1): p. 7-14. 39. Hauck, C.R., et al., Cellular adhesion molecules as targets for bacterial infection. Eur J Cell Biol, 2006. 85(3-4): p. 235-42. 40. Boyle, E.C. and B.B. Finlay, Bacterial pathogenesis: exploiting cellular adherence. Curr Opin Cell Biol, 2003. 15(5): p. 633-9. 41. Hornick, D.B., et al., Fimbrial types among respiratory isolates belonging to the family Enterobacteriaceae. J Clin Microbiol, 1991. 29(9): p. 1795-800. 42. Favre-Bonte, S., A. Darfeuille-Michaud, and C. Forestier, Aggregative adherence of Klebsiella pneumoniae to human intestine-407 cells. Infect Immun, 1995. 63(4): p. 1318-28. 43. Hornick, D.B., et al., Adherence properties of an mrkD-negative mutant of Klebsiella pneumoniae. Infect Immun, 1995. 63(5): p. 2026-32. 44. Tarkkanen, A.M., et al., Binding of the type 3 fimbriae of Klebsiella pneumoniae to human endothelial and urinary bladder cells. Infect Immun, 1997. 65(4): p. 1546-9. 45. Fung, C.P., et al., Klebsiella pneumoniae in gastrointestinal tract and pyogenic liver abscess. Emerg Infect Dis, 2012. 18(8): p. 1322-5. 46. Hsu, C.R., et al., Klebsiella pneumoniae translocates across the intestinal epithelium via Rho GTPase- and phosphatidylinositol 3-kinase/Akt-dependent cell invasion. Infect Immun, 2015. 83(2): p. 769-79. 47. de Lorenzo, V., et al., Mini-Tn5 transposon derivatives for insertion mutagenesis, promoter probing, and chromosomal insertion of cloned DNA in gram-negative eubacteria. J Bacteriol, 1990. 172(11): p. 6568-72. 48. Chun, K.T., et al., Rapid amplification of uncharacterized transposon-tagged DNA sequences from genomic DNA. Yeast, 1997. 13(3): p. 233-40. 49. Link, A.J., D. Phillips, and G.M. Church, Methods for generating precise deletions and insertions in the genome of wild-type Escherichia coli: application to open reading frame characterization. J Bacteriol, 1997. 179(20): p. 6228-37. 50. Raffel, F.K., et al., The Haemophilus influenzae Sap transporter mediates bacterium-epithelial cell homeostasis. Infect Immun, 2013. 81(1): p. 43-54. 51. Cruz, D.P., et al., Comparative analysis of diguanylate cyclase and phosphodiesterase genes in Klebsiella pneumoniae. BMC Microbiol, 2012. 12: p. 139. 52. Mills, E., et al., The bacterial second messenger c-di-GMP: mechanisms of signalling. Cell Microbiol, 2011. 13(8): p. 1122-9. 53. Hong, S.H., X. Wang, and T.K. Wood, Controlling biofilm formation, prophage excision and cell death by rewiring global regulator H-NS of Escherichia coli. Microb Biotechnol, 2010. 3(3): p. 344-56. 54. Castang, S., et al., H-NS family members function coordinately in an opportunistic pathogen. Proc Natl Acad Sci U S A, 2008. 105(48): p. 18947-52. 55. Beachey, E.H., Bacterial adherence: adhesin-receptor interactions mediating the attachment of bacteria to mucosal surface. J Infect Dis, 1981. 143(3): p. 325-45. 56. Coutte, L., et al., Role of adhesin release for mucosal colonization by a bacterial pathogen. J Exp Med, 2003. 197(6): p. 735-42. 57. Maroncle, N., et al., Identification of Klebsiella pneumoniae genes involved in intestinal colonization and adhesion using signature-tagged mutagenesis. Infect Immun, 2002. 70(8): p. 4729-34. 58. Spurbeck, R.R., R.J. Tarrien, and H.L. Mobley, Enzymatically active and inactive phosphodiesterases and diguanylate cyclases are involved in regulation of Motility or sessility in Escherichia coli CFT073. MBio, 2012. 3(5). 59. Struve, C., M. Bojer, and K.A. Krogfelt, Characterization of Klebsiella pneumoniae type 1 fimbriae by detection of phase variation during colonization and infection and impact on virulence. Infect Immun, 2008. 76(9): p. 4055-65. 60. Miller, V.L. and J.J. Mekalanos, A novel suicide vector and its use in construction of insertion mutations: osmoregulation of outer membrane proteins and virulence determinants in Vibrio cholerae requires toxR. J Bacteriol, 1988. 170(6): p. 2575-83. 61. Herrero, M., V. de Lorenzo, and K.N. Timmis, Transposon vectors containing non-antibiotic resistance selection markers for cloning and stable chromosomal insertion of foreign genes in gram-negative bacteria. J Bacteriol, 1990. 172(11): p. 6557-67. 62. Salama, N.R., B. Shepherd, and S. Falkow, Global transposon mutagenesis and essential gene analysis of Helicobacter pylori. J Bacteriol, 2004. 186(23): p. 7926-35. | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/54069 | - |
| dc.description.abstract | 克雷伯氏肺炎桿菌(Klebsiella pneumoniae)為一種伺機性感染的病原菌，常引起肺炎、敗血症、泌尿系統發炎及社區性肝膿瘍等感染。細菌附著於宿主細胞通常是達成感染的第一步驟，因此黏附因子(adhesin)可能在細菌貼附到細胞的過程中扮演重要角色。克雷伯氏肺炎桿菌為腸道中的正常菌叢，最近我們的研究也顯示在腸道中克雷伯氏肺炎桿菌會穿過(translocation)腸道上皮而造成感染，因此我們想找尋克雷伯氏肺炎桿菌與腸道上皮細胞貼附相關的基因。利用跳躍子(transposon)建構了克雷伯氏肺炎桿菌血清型K2菌株Ca0437之突變株庫(mutant library)，此突變株庫包含4600株的突變株，以Caco-2細胞作黏附試驗(adherence assay)，一共篩選了2450株突變株，發現其中有9株突變菌株其黏附能力有明顯的下降，以NCBI-BLAST作基因功能比對分析，這些被跳躍子破壞的基因可分為含有跨膜區域(transmembrane domain)的基因、調控(regulator)基因、酵素(enzyme)與其它(others)。利用無標誌基因剔除法(unmarked gene deletion)方式分別建構sapA、dgc與hns基因剔除菌株，驗證這些基因剔除菌株之細胞黏附能力下降，且細菌生長試驗證實非因生長差異所致。也建構了相對之染色體互補(chromosome complementation)菌株，發現dgc與hns基因染色體互補菌株的細胞黏附能力有補回，而sapA基因剔除菌株則是利用質體補回操縱子sapABC使得其細胞黏附能力回復。此外，hns為一個上游之調控基因，進一步利用基因微陣列分析其調控克雷伯氏肺炎桿菌之基因轉錄，發現可影響第一型線毛的相關基因表現。分別以胃內感染和腹膜注射感染小鼠的方式評估sapA、dgc與hns基因剔除株對於致病力(virulence)的影響，發現在胃內感染的情況下，hns基因剔除株的致病力較野生株有明顯下降，其餘的基因剔除菌株則與野生株並無顯著差異，因此未來會對基因剔除株的致病力進行實驗來作進一步探討。 | zh_TW |
| dc.description.abstract | Klebsiella pneumoniae is an opportunistic pathogen associated with pneumonia, septicemia, urinary tract infection, pyogenic liver abscess and other infections. Bacterial adherence to host cells is usually the first step in pathogenesis of infections, and therefore adhesins play an important role in the process. K. pneumoniae is the normal intestinal flora, and recent studies showed translocation of K. pneumoniae through the intestinal epithelial cells leading to dissemination of the infection into the bloodstream. Hence, we wanted to explore the genes responsible for adhesion to intestinal epithelial cells in K. pneumonia. We constructed the transposon mutant library of K. pneumonia serotype K2 strain Ca0437, and the library included 4600 mutants. The mutant library was screened by adherence assay with Caco-2 cells. A total of 2450 mutants were screened and nine mutants decreased in the ability of adhesion to cells. Through NCBI-BLAST for gene function analysis, the gene inserted by transponsons can be divided into the genes with transmembrane domain, regulatory genes, enzymes and others. Unmarked deletion and complementation of sapA, dgc and hns gene demonstrated that they were responsible for adhesion to cells, instead of the differences of bacterial growth rate. In order to study the role of gene regulation in hns gene, we compared the RNA expression profiles of Ca0437Δhns with those of Ca0437 wild-type by microarray. The two clones which expressions were down-regulated in Ca0437Δhns contained genes in association with type 1 fimbriae. In vivo experiments showed that intragastric infection of Δhns mutant had significant differences with the wild-type strain. And we will study the virulence of the other deletion mutants in the future. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-16T02:38:45Z (GMT). No. of bitstreams: 1 ntu-104-R02445109-1.pdf: 2364790 bytes, checksum: 9816ec78164eb197c5100b1ab3553577 (MD5) Previous issue date: 2015 | en |
| dc.description.tableofcontents | 口試委員審定書…………………………………………………………………………I 致謝……………………………………………………………………………………...II 中文摘要……………………………………………………………………………….III 英文摘要…………………………………………………………………………….....IV 目錄………………………………………………………………………………….... VI 表目錄………………………………………………………………………………..VIII 圖目錄………………………………………………………………………………….IX 第一章、緒論………………………………………………………………………….…1 1. 克雷伯氏肺炎桿菌………………………...……….…………..…………..1 2. 克雷伯氏肺炎桿菌與疾病……………..………………………………....1 3. 克雷伯氏肺炎桿菌的主要致病因子…………………………..………….2 4. 克雷伯氏肺炎桿菌造成感染致病的首要步驟：黏附作用..……………..3 5. 研究目的.…………………………………………………………………...3 第二章、材料與方法…………………………………………………………………….5 1. 菌株與載體……………………………………………..……..…………..5 2. 建立克雷伯氏肺炎桿菌突變株庫………………………..……………....5 3. 突變株序列分析……………………………………….………………….6 4. 細胞黏附試驗……………………………………………………………..8 5. 建構克雷伯氏肺炎桿菌基因剔除株.…………………………..………...9 6. 建構突變株的染色體互補……………………..……….………………...10 7. 建構 sapABC 基因操作子的異位互補………..……………….……......12 8. 細菌生長曲線測量…………………..…………………………………....12 9. 克雷伯氏肺炎桿菌total RNA 抽取……………………………….……..12 10. 克雷伯氏肺炎桿菌基因微陣列………………………….……………13 11. 反轉錄定量聚合酶鏈鎖反應（RT-qPCR）…………………..…….……17 12. 動物實驗…………………………………………..…………………..…18 第三章、結果…………………………………………………………………………...20 1. 建構克雷伯氏肺炎桿菌突變株庫………………………………….……20 2. 突變株多樣性測試…………………………….…………………….......20 3. 利用細胞黏附試驗篩選克雷伯氏肺炎桿菌突變株庫……..……………20 4. 突變株序列分析並確認Ca0437菌株所被突變的基因…..……...………21 5. 建構克雷伯氏肺炎桿菌基因剔除突變株與基因補回菌株………….….22 6. 利用克雷伯氏肺炎桿菌基因微陣列尋找hns所影響的基因…………....24 7. 動物感染實驗……………….…………………………………………….24 第四章、總結與討論…………………………………………………………………...25 第五章、參考文獻……………………………………………………………………...56 附錄一、利用NCBI-BLAST進行比對分析出跳躍基因在突變株中插入的位置….61 表目錄 表一、研究使用之菌株及載體……………………………………………………… 29 表二、實驗使用之引子………………………………………………………………..30 表三、利用細胞黏附試驗篩選克雷伯氏肺炎桿菌突變株庫結果…………….…….32 表四、利用克雷伯氏肺炎桿菌基因微陣列工具，以Ca0437野生株為對照組、Ca0437Δhns突變株為實驗組，比較兩者基因的RNA表現量………..…....33 圖目錄 圖一、半隨機聚合酶鏈鎖反應(semi-random PCR)…………………………..…….34 圖二、以細胞黏附試驗所找出黏附能力下降突變株…..……….…………..,,,……...35 圖三、pKO3-Km基因剔除載體及突變株篩選過程……………….………………...36 圖四、利用PCR確定Ca0437ΔsapA突變菌株…………………..…………………..37 圖五、利用PCR確定Ca0437Δdgc突變菌株……..……………………….......……..38 圖六、利用PCR確定Ca0437Δhns突變菌株………………………………...………39 圖七、野生型菌株與突變株的生長曲線……...............................................................40 圖八、利用細胞黏附試驗比較Ca0437野生株、Ca0437 P9-10E突變株、Ca0437ΔsapA 突變株………………………………………………………………………….41 圖九、利用細胞黏附試驗比較Ca0437野生株、Ca0437 P11-2A突變株、Ca0437Δdgc突變株.……………….……………………………………..........……...…...42 圖十、利用細胞黏附試驗比較Ca0437野生株、Ca0437 P9-6E突變株、Ca0437Δhns突變株………………………………………………………………………..43 圖十一、pKO3-Km基因互補載體及染色體互補株篩選過程….…………..……...…44 圖十二、利用PCR確定Ca0437ΔsapA::sapA染色體互補菌株……………………...45 圖十三、利用PCR確定Ca0437Δdgc::dgc染色體互補菌株………………….........46 圖十四、利用PCR確定Ca0437Δhns::hns染色體互補菌株……………...……….47 圖十五、利用細胞黏附試驗比較Ca0437野生株、Ca0437ΔsapA突變株、Ca0437ΔsapA::sapA染色體互補菌株..…………………...….........………..48 圖十六、利用細胞黏附試驗比較Ca0437野生株、Ca0437Δdgc突變株、Ca0437Δdgc::dgc染色體互補菌株…….………………….……………..…49 圖十七、利用細胞黏附試驗比較Ca0437野生株、Ca0437Δhns突變株、Ca0437Δhns::hns染色體互補菌株……………….…...…………………….50 圖十八、以RT-qPCR檢視sapA、sapB、sapC於Ca0437野生株、Ca0437ΔsapA突變株、Ca0437ΔsapA::sapA染色體互補菌株的表現情形…………….…….51 圖十九、利用細胞黏附試驗比較Ca0437野生株、Ca0437ΔsapA突變株、Ca0437ΔsapA::sapABC質體互補菌株…………………………………………..…52 圖二十、利用克雷伯氏肺炎桿菌基因微陣列工具，以Ca0437野生株為對照組、Ca0437Δhns突變株為實驗組，比較兩者基因的RNA表現量……………53 圖二十一、突變株與野生株以胃內感染方式感染小鼠的存活情形…………...……54 圖二十二、突變株與野生株以腹腔注射方式感染小鼠的存活情形……………..….55 | |
| dc.language.iso | zh-TW | |
| dc.title | 從突變基因庫找出克雷伯氏肺炎桿菌參與腸道細胞黏附作用相關的基因 | zh_TW |
| dc.title | Isolation of Klebsiella pneumoniae gene(s) involved in adhesion to intestinal epithelial cells using a mutant library | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 103-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 楊宏志(Hung-Chih Yang),蔡丰喬(Feng-Chiao Tsai),林雅容 | |
| dc.subject.keyword | 克雷伯氏肺炎桿菌,突變株庫,黏附因子,致病力, | zh_TW |
| dc.subject.keyword | Klebsiella pneumoniae,mutant library,adhesin,virulence, | en |
| dc.relation.page | 61 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2015-07-23 | |
| dc.contributor.author-college | 醫學院 | zh_TW |
| dc.contributor.author-dept | 微生物學研究所 | zh_TW |
| 顯示於系所單位： | 微生物學科所 | |
文件中的檔案：
| 檔案 | 大小 | 格式 | |
|---|---|---|---|
| ntu-104-1.pdf 目前未授權公開取用 | 2.31 MB | Adobe PDF |
系統中的文件，除了特別指名其著作權條款之外，均受到著作權保護，並且保留所有的權利。