臺灣博碩士論文加值系統

摘要
Klebsiella pneumonia 是人體腸道的常在菌，但在免疫低下的病人常造成疾病。近年來，由於院內感染Klebsiella pneumonia所導致的肝膿瘍更是常見。有研究指出，在國內因感染Klebsiella pneumonia所導致肝膿瘍的病患中，約有75﹪的病患同時伴隨著type Π DM。實驗發現，由肝膿瘍病患所分離的Kp5菌株，其cps region上共有4個具有啟動子活性的片段。其中位於orf2和wzi這兩個 open reading frames(ORFS)之間的啟動子B，活性在受到葡萄糖誘導後會大幅上升，其後的基因wzi之mRNA表現量也有明顯的增加。研究啟動子B之後基因wzi所轉譯出來的蛋白是否具有調控下游啟動子G之作用，結果發現下游的啟動子G，在啟動子B及wzi基因共同存在下，其活性會上升。在葡萄糖誘導下，啟動子G的活性也比單獨測得時來得高一些，但其後基因gmd、wcaG的表現量未增加，我們推測可能與葡萄糖溶液誘導下，莢膜中大量表現fucose而產生feedback inhibition有關。另外我們也發現，將Kp5菌種培養在含有葡萄糖溶液的LB broth中，其生長速率比養在未含有葡萄糖的LB broth中來的快。因此我們推測，細菌利用代謝葡萄糖產生能量，促進其本身生長繁殖之速率，及病患本身免疫力低下，是造成此菌對糖尿病患有高感染率的主要原因。

Abstract

Klebsiella pneumoniae is a normal flora present in human intestinal tract, but will cause disease in immunocompromised patients. Primary pyogenic liver abscess (PLA) caused by Klebsiella pneumoniae is an emerging infectious disease especially in diabetic patients. The effect of glucose concentrations on the promoter of capsular polysaccharide (cps) gene region was investigated. The putative promoters of the Kp5, a virulent strain, were cloned into a plasmid containing -galactosidase reporter system. Seven putative promoter regions, designated A, B, C, D, E, F, G, were cloned and the -galactosidase activity assays suggested that the regions of galF to orf2（A）, orf2 to wzi（B）, wzi to wza（G）, and rfbp to gnd（E）have promoter activities. The promoter B in the upstream region of wzi showed higher promoter activity than others, enhanced by glucose addition, and wzi mRNA expression was elevated at the same time. To study transcription regulation of gene wzi, we observed that the activity of promoter G was elevated under the influence of promoter B and wzi gene. The over all activity of promoter G was increased under high glucose condition, however the gmd and wcaG mRNA expression was decreased, suggesting that the decrease of gmd and wcaG mRNA expression might be due to the feedback inhibition of the increased fucose final product. We also observed that Kp5 increased its growth rate at high glucose condition. In summary, our results suggested that high glucose condition, such as in the case of diabetes enhanced bacterial growth and the immunocompromised state of the DM patients were the major cause of diabetic patients with high susceptibility to Klebsiella pneumoniae infection.

目 錄
指導教授推薦書
口試委員會審定書
授權書………………………………………....................................iii
致謝……………………………………………….............................iv中文摘要........................………………………….…......................v
英文摘要……………………………………..……...........................vi
目錄………………………………………………...........................viii
圖表目錄......................................................................................ix
縮寫表...........................................................................................x
第一章 緒論............................................................................................1
第二章 研究動機.............................…………................................9
第三章 材料方法……………………….........................................11
第四章 實驗結果..................................………………..................20
第五章 實驗討論...........................…...........................................26
第六章 圖表................................................................................32
參考文獻......................................................................................48


圖表目錄
圖一：KP NTUH-k204菌株之莢膜合成相關基因組…...................4
圖二：KP致病相關毒力因子………………………………............32圖三：-galactosidase活性測定確定可能的promoter位置............33
圖四：不等濃度之葡萄糖溶液誘導 -galactosidase活性的改變......35 圖五：未做real time PCR前之RT-PCR檢視gmd、wcaG及wzi基因表現.........................................................................................37
圖六：Real time PCR分析環境中0mg/dl和400mg/dl葡萄糖溶液對gmd、wcaG及wzi基因表現的差異…………………………………38
圖七：promoter B與wzi基因共同調控下的promoter G之-galactosidase活性的改變……...................................................40
圖八：Kp5於環境中外加0mg/dl和400mg/dl葡萄糖溶液之生長曲線與活菌數…………………………………………….....................42
圖九：pKM005的限制酶圖譜.......................................................44
表一：本實驗所使用之菌種及質體................................................45
表二：本實驗所使用之單股核酸引子.............................................47

文獻探討
1. Podschun, R. and Ullmann, U. 1998. Klebsiella spp. as nosocomial pathogens: epidemiology, taxonomy, typing methods, and pathogenicity factors. Clin Microbiol Rev 11(4): 589-603.
2. Yi-Pang Chuang, Chi-Tai Fang, Shau-Yan Lai, Shan-Chwen Chang, Jin-Town Wang. Genetic Determinants of Capsular Serotype K1 of Klebsiella Pneumoniae Causing Primary Pyogenic Liver Abscess. Journal of Infectious Disease . 2006；193：645-54
3. Greer E. Kaufman, Janet Yother. CcpA-depedent and independent control of beta-glactosidase expression in Streptococcus pneumoniae occurs via regulation of an upstream PTS-encoding operon. Journal Bacteriology. 2007；doi：10.1128
4. Laakso, D. H., M. K. Homonylo, S. J. Wilmot, and C. Whitfield. 1988. Transfer and expression of the genetic determinants for O and K antigen synthesis in Escherichia coli O9:K(A)30 and Klebsiella sp. O1:K20 in Escherichia coli K12. Can. J. Microbiol. 34:987-992
5. Matsumoto, H., and T. Tazaki. 1971. Genetic mapping of aro, pyl, and pur markers in Klebsiella pneumoniae. Jpn. J. Microbiol. 15:11-20
6. Drummelsmith, J., and Whitfield, C. 2000. Translocation of group 1 capsular polysaccharide to the surface of Escherichia coli requires a multimeric comple in the outer membrane. EMBO J 19:57-66
7. Wugeditsch, T., Paiment, A., Hocking, J., Drummelsmith, J., Forrester, C., and Whitfield, C. 2001. Phosphorylation of Wzc, a tyrosine autokinase, is essential for assembly of group 1 capsular polysaccharides in Escherichia coli. J Biol Chem 276:2361-2371
8. Clarke, D.J., Joyce, S.A., Toutain, C.M., Jacq, A., and Holland, I.B. 2002. Genetic analysis of the RcsC sensor kinase from Escherichia coli K-12. J Bavteriol 184:1204-1208
9. Gottesman, S. 1995. Regulation of capsule synthesis : modification of the two- component paradigm by an accessory unstable regulator. In: Two-component Signal Transduction. Hoch, J.A., and Silhavy, T.J. （eds）. Washington, DC: American Society for Microbiology Press, pp. 253-262
10. Stout, V. 1994. Regulation of capsule synthesis includes interaction of the RcsC/RcsB regulatory pair. Res Microbiol 145:389-392
11. Takeda, S., Fujisawa, Y., Matsubara, M., Aiba, H., and Mizuno, T.2001. A novel feature of the multistep phosphorelay in Escherichia coli: a revised model of the RcsC－＞YojN－＞RcsB signaling pathway implicated in capsular synthesis and swarming behaviour. Mol Microbiol 40:440-450
12. Arakawa, Y., Wacharotayankun, R., Nagatsuka, T., Ito, H., Kato, N., and Ohta, M. 1995.Genomic organization of the Klebsiella pneumoniae cps region rewponsible fpr serotype K2 capsular polysaccharides synthesis in the virulent strain Chedid. J Bacteriol 177:1788-1796
13. Rahn, A., Drummelsmith, J., and Whitfield, C. 1999. Conserved organization in the cps gene cluster for epression of Escherichia coli group 1 K antigens: relationship to the colanic acid biosynthesis locus and the cps genes from Klebsiella pneumoniae. J Bacteriol 181: 2307-2313
14. Stout, V., A. Torres-Cabassa, M. R. Maurizi, D. Gutnick, and S. Gottesman. 1991. RcsA, an unstable positive regulator of capsular polysaccharide synthesis. J. Bacteriol. 173:1738-1747
15. Yi-Chyi Lai, Hawei-Ling Peng, and Hwan-You Chang. 2003. RmpA2, an activator of capsule biosynthesis in Klebsiella pneumoniae CG43, regulates K2 cps gene expression at the transcription level. J Bacteriol 185:788-800
16. Kolb, A., S. Busby, H. Buc, S. Garges, and S. Adhya. 1993. Transcriptional regulation by cAMP and its receptor protein. Annu. Rev. Biochem. 62:749-795
17. Zheng, D., C. Constantinidou, J. L. Hobman, and S.D. Minchin. 2004. Identification of the CRP regulon using in vitro transcriptional profiling. Nucleic Acids Res. 32:5874-5893
18. Heyduk, T., and J. C. Lee. 1989. Escherichia coli cAMP receptor protein : evidence for three protein conformational states with different promoter binding affinities. Biochemistry 28:6914-6924
19. Mckay, D. B., and T. A.Steitz. 1981. Structure of catabolite gene activator protein at 2.9À resolution suggests binding to left-handed B-DNA. Nature 290:744-749
20. Won, H. S., T. W. Lee, S. H. Park, and B. J. Lee. 2002. Stoichiometry and structural effect of the cyclic nucleotide binding to cyclic AMP receptor protein. J. Biol. Chem. 277:11450-11455
21. Won, H. S., T. W. Yamazaki, T. W. Lee, M. K. Yoon, S. H. Park, Y. Kyogoku, and B. J. Lee. 2000. Structural understanding of the allosteric conformational change of cyclic AMP receptor protein by cyclic AMP binding. Bilchemistry 39:13953-13962
22. Ebright, R. H., Y. W. Ebright, and A. Gunasekera. 1989. Consensus DNA site for the Escherichia coli catabolite gene protein（CAP）: CAP exhibits a 450-gold higher affinity for the consensus DNA sites than for the E. coli lac DNA site. Nucleic Acids Res. 17:10295-10305
23. Botsford, J. L., and J. G. Harman. 1992. Cyclic AMP in prokaryotes. Microbiol. Rev. 56:100-122
24. Sung Kuk Lee and Jay D. Keasling. 2006. Effect of glucose as the sole carbon source on gene expression from the Salmonella prpBCDE promoter in Escherichia coli. 22:1547-1551
25. Yih-Ling Tzeng, John S. Swartley, Yoon K. Miller, Rachel E. Nisbet, Li-Jun Liu, Jay H. Ahn, and David S. Stephens. 2001. Transcription regulation of divergent capsule biosynthesis and transport operon promoters in serogroup B Neisseria meningitides
26. M. Stoecklea, C. Kaechb, A. Trampuzb, W. Zimmerlia. 2008. The role of diabetes mellitus in patientswith bloodstream infections. SWISS MED WKLY 138:512–519
27. Drivas G, Wardle N. 1978. Reticuloendothelial cell dysfunction in diabetes and hyperlipidemia. 27:1533-8
28. Wolf G, Müller N, Busch M, Eidner G, Kloos C, Hunger-Battefeld W, Müller UA. 2009. Diabetic foot syndrome and renal function in type 1 and 2 diabetes mellitus show close association.
29. Boris Görke & Jörg Stülke. 2008. Carbon catabolite repression in bacteria: many ways to make the most out of nutrients. Nature Reviews Microbiology. 6: 613-624
30. Melinda Pitt, Maria-Trinidad Gallegos and Martin Buck. 2000. Single amino acid substitution mutants of Klebsiella pneumoniaeσ54 defective in transcription. Nucleic acids research. 22:4419-4427
31. Siva R. Wigneshwerara, Sergei Nechaev, Konstantin Severinov and Martin Buck. 2002. Beta subunit residues 186-433 and 436-445 are commonly used by Eσ54 and Eσ70 RNA polymerase for open promoter complex formation. J. Mol. Biol. 319:1067-1083