跳到主要內容

臺灣博碩士論文加值系統

(18.97.14.82) 您好!臺灣時間:2025/02/15 02:03
字體大小: 字級放大   字級縮小   預設字形  
回查詢結果 :::

詳目顯示

: 
twitterline
研究生:萬舉豪
研究生(外文):Wan, Chu-Hao
論文名稱:克雷白氏肺炎桿菌CG43中磷酸甘露糖異構酶磷酸化的特性探討
論文名稱(外文):Characterization of the ManB phosphorylation in Klebsiella pneumoniae CG43
指導教授:彭慧玲彭慧玲引用關係
指導教授(外文):Peng, Hwei-Ling
學位類別:碩士
校院名稱:國立交通大學
系所名稱:生物科技學系
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2012
畢業學年度:100
語文別:中文
論文頁數:64
中文關鍵詞:克雷白氏肺炎菌磷酸化
外文關鍵詞:Klebsiella pneumoniaephosphorylation
相關次數:
  • 被引用被引用:1
  • 點閱點閱:181
  • 評分評分:
  • 下載下載:20
  • 收藏至我的研究室書目清單書目收藏:0
細菌酪胺酸激酶是近年來新建立的蛋白質家族,與真核生物之酪胺酸激酶有著
相似的功能。活體外磷酸化實驗證明克雷白氏肺炎菌 CG43 之酪胺酸激酶 Wzc
可以將多醣莢膜基因座上之基因產物包括 6-葡萄醣醛酸脫氫酶 Gnd、甘露糖-1-
磷酸鳥苷醯基轉移酶 ManC 及磷酸甘露糖異構酶 ManB 磷酸化,並增加 Gnd 之
酵素活性。本實驗建構克雷白氏肺炎菌 CG43 突變株 Δgnd、ΔmanC 與 ΔmanB
並分析基因缺失的影響,結果發現 ΔmanC 與 ΔmanB 之多醣莢膜合成量明顯下降而生物膜生成增加,為了證實 ManB 活性與酪胺酸殘基磷酸化的關係,我們藉由比對分析選出可能受磷酸化的酪胺酸殘基做定點突變,接著回補實驗分析發現
manB 基因剔除所造成的多醣莢膜含量下降可藉轉殖 ΔmanB 予表現質體
pRK415-manB、pRK415- manBY26F 或 pRK415-manB Y341F 而回補。然而
pRK415-manBY10F 僅能回復些微多醣莢膜含量,此暗示著 Tyr10 可能影響 ManB
之活性。而分析全菌裂解液之 ManB 酵素活性發現 Y10F、Y26F 及 Y341F 突變
會降低 ManB 酵素活性而以 Y10F 下降程度最大;另外,S98A 突變使 ManB 喪
失活性。進一步,在大腸桿菌中大量表現這些重組蛋白,以圓雙色光譜分析這些
純化的重組蛋白證實這些酪胺酸殘基突變並不影響 ManB 之二級結構,我們推測
Y10F 可能因降低 ManB 與受質的結合力而影響其催化活性,而 S98 的磷酸化決
定其酵素活性。最後,活體外磷酸化實驗顯示單株抗體和 Pro-Q Diamond 均可偵測 ManB 的磷酸化,ManB 的磷酸化些微提升其酵素活性;而 S98A 突變使
ManB 不被磷酸化且活性喪失,而其磷酸化不受酪胺酸激酶或絲胺酸-蘇胺酸激酶
的基因缺損影響。
Bacterial tyrosine kinases have been recently unified in a new enzyme family because of the similar structural and functional features with their eukaryotic counterparts. Via an in vitro phosphorylation assay, the protein-tyrosine kinase Wzc of Klebsiella pneumoniae CG43 was shown capable of phosphorylating the capsular polysaccharide biosynthesis operon cps gene products 6-phosphogluconate
dehydrogenase (Gnd), GDP-mannose phosphorylase (ManC) and
phosphomannomutase (ManB) and the phosphorylation leading to increase of the enzymatic activities. In this study, we generate CG43S3Δgnd, CG43S3ΔmanC and CG43S3ΔmanB mutants and the phenotypes analyzed and compared. The analysis revealed that deletion of manC or manB decreased the glucuronic acid production but increased the biofilm formation. Several of the ManB tyrosine residues were then selected for site directed mutagensis in order to determine which is the one that subjected to Wzc phosphorylation and if the phosphorylation influences the enzymztic activity. The subsequent complementation analysis revealed that the manB deleting effect could be complemented by introducing into the mutant with the ManB expression plasmid pRK415-manB or the plasmid pRK415-manBY26F and pRK415-manBY341F which carrying a point mutation of ManB.Whereas the bacteria CG43S3ΔmanB carrying pRK415-manBY10F exhibited a partially restored phenotype implying the phosphorylation on tyrosine residue 10 of ManB was critical for the enzymatic activity. In addition, the ManB activity was decreased in the bacteria CG43S3ΔmanB [pETQ33-manBY10F] or CG43S3ΔmanB [pETQ33-manBY341F] by comparing to that in CG43S3ΔmanB [pETQ33-manB ] or CG43S3ΔmanB [pETQ33- manBY26F]. These recombinant plasmids were then overexpressed in E. coli BL21[DE3] and the recombinant proteins purified. The circular dichroism spectra analysis of the purified proteins revealed that the site directed mutation had no effect on the secondary structure. Finally, the in virto phosphorylation assay showed that the monoclonal antibody and Pro-Q Diamond were able to detect the ManB phosphorylation form and ManB phosphorylation slightly enhanced the enzymatic activity. In addition, the S98 was found to be a critical phosphorylation residue which is required for the enzyme activity. However,the serine phosphorylation was not afftected by the deletion of wzc or stk.
中文摘要…………………………………………………………………i
英文摘要…………………………………………………………………ii
致謝………………………………………………………………………iv
目錄………………………………………………………………………vi
表目錄……………………………………………………………………vii
圖目錄……………………………………………………………………viii
縮寫………………………………………………………………………x
前言………………………………………………………………………1
實驗材料與方法…………………………………………………………8
實驗結果…………………………………………………………………15
討論………………………………………………………………………21
相關文獻…………………………………………………………………24
表…………………………………………………………………………31
圖…………………………………………………………………………37
附錄………………………………………………………………………61

1.白平輝.(2004).克雷白氏肺炎桿菌 CG43 莢膜多醣體的產生和酪胺酸磷酸化作用的研究. 碩士論文,生物科技所,國立交通大學.
2.李智凱.(2006).克雷白氏肺炎桿菌CG43中參與莢膜多醣體生合成之核心蛋白Wza、Yor5、Yco6 和Wzx的功能性研究.碩士論文,生物科技所,國立交通大學.
3.錢漢聲. (2008).克雷白氏肺炎桿菌酪胺酸激酶KpWzc對其下游酵素ManC, Gnd活性影響之分析與Ugd磷酸酪胺酸殘基鑑定與下游受質之搜尋.碩士論文,分子醫學所,國立清華大學
4.Adams, M. J., Ellis, G. H., Gover, S., Naylor, C. E., and Phillips, C. (1994) Crystallographic study of coenzyme, coenzyme analogue and substrate binding in 6-phosphogluconate dehydrogenase: implications for NADP specificity and the enzyme mechanism. Structure 2, 651-668.
5.Brisse S, Grimont F, Grimont PAD. (2006). The genus Klebsiella. In: Dworkin M,Falkow S, Rosenberg E, Schleifer K-H, Stackebrandt E, eds. The Prokaryotes. AHandbook on the Biology of Bacteria. 3rd edition ed. New York: Springer.
6.Chang, H.Y., Lee, J.H., Deng, W.L., Fu, T.F., and Peng, H.L. (1996) Virulence and outer membrane properties of a galU mutant of Klebsiella pneumoniae CG43. Microb Pathog 20: 255-261.
7.Coligan, J.E. (2003) Current Protocols in Protein Science, John Wiley & Sons Inc., Brooklyn, NY, Unit 7.6.
8.Coyne, M.J., Jr, Russell, K.S., Coyle, C.L., and Goldberg, J.B. (1994) The Pseudomonas aeruginosa algC gene encodes phosphoglucomutase, required for the synthesis of a complete lipopolysaccharide core. J Bacteriol 176:3500–3507.
9.Cozzone, A. J. (2005) Role of protein phosphorylation on serine/threonine and tyrosine in the virulence of bacterial pathogens. J Mol Microbiol Biotechnol 9, 198-213.
10.Cozzone, A. J. (2009) Bacterial tyrosine kinases: novel targets for antibacterial therapy? Trends Microbiol. 17 536–543
11.Domenico, P., Schwartz, S., and Cunha, B.A. (1989) Reduction of capsular polysaccharide production in Klebsiella pneumonia by sodium salicylate. Infect Immun 57: 3778-3782.
12.Dong C, Beis K, Nesper J, Brunkan-Lamontagne AL, Clarke BR, et al. (2006). Wza the translocon for E. coli capsular polysaccharides defines a new class of membrane protein. Nature 444:226–29
13.Dower, W.J., Miller, J.F., and Ragsdale, C.W. (1988) High efficiency transformation of E. coli by high voltage electroporation. Nucleicd Acids Res 16: 6127-6145.
14.Fantl WJ, Johnson DE, Williams LT. (1993). Signalling by receptor tyrosine kinases. Annu Rev Biochem. 62:453-81.
15.Garnak M, Reeves HC (1979) Phosphorylation of isocitrate dehydrogenase of Escherichia coli. Science 203:1111–1112
16.Goldberg J. B., Hatano K., Pier G. B. (1993). Synthesis of lipopolysaccharide O side chains by Pseudomonas aeruginosa PAO1 requires the enzyme phosphomannomutase. J. Bacteriol. 175, 1605–1611.
17.Grangeasse C, Doublet P, Cozzone AJ. (2002). Tyrosine phosphorylation of protein kinase Wzc from Escherichia coli K12 occurs through a two-step process. J Biol Chem. 277(9):7127-35.
18.Grangeasse C, Obadia B, Mijakovic I, Deutscher J, Cozzone A J and Doublet P. (2003) Autophosphorylation of the Escherichia coli protein kinase Wzc regulates tyrosine phosphorylation of Ugd, a UDP-glucose dehydrogenase. J Biol Chem 278: 39323–39329.
19.Grangeasse C, Cozzone AJ, Deutscher J, Mijakovie I. (2007) Tyrosine phosphorylation: an emerging regulatory device of bacterial physiology. Trends Biochem Sci. 32(2):86-94
20.Hanau, S., Montin, K., Cervellati, C., Magnani, M. and Dallocchio, F.(2010) 6-Phosphogluconate dehydrogenase mechanism: evidence for
allosteric modulation by substrate. J. Biol. Chem. 285, 21366–21371.
21.Ho J-Y, Lin T-L, Li C-Y, Lee A, Cheng A-N, Chen M-C, Wu S-H, Wang J-T, Li T-L, Tsai M-S. (2011) Functions of Some Capsular Polysaccharide Biosynthetic Genes in Klebsiella pneumoniae NTUH K-2044. PLoS ONE 6(7):
22.Hunter T (2000) Signaling—2000 and beyond. Cell 100:113–127
23.Henriques, M. X., Rodrigues, T., Carido, M., Ferreira, L. and Filipe, S. R. (2011), Synthesis of capsular polysaccharide at the division septum of Streptococcus pneumoniae is dependent on a bacterial tyrosine kinase. Molecular Microbiology, 82: 515–534.
24.Joanna B. Goldberg, Kazue Hatano, and Gerald B. Pier. (1993). Synthesis of Lipopolysaccharide 0 Side Chains by Pseudomonas aeruginosa PAO1 Requires the Enzyme Phosphomannomutase. J Bacteriol. 1993 March; 175(6): 1605–1611.
25.Keen, N.T., Tamaki, S.,Kobayashi, D. and Trollinger, D. (1988) Gene 70, 191- 197.
26.Kelly S.M., Price N.C. (1997) The application of circular dichroism to studies of protein folding and unfolding. Biochim Biophys Acta 1338:161–185.
27.Lacour S, Bechet E, Cozzone AJ, Mijakovic I, Grangeasse C (2008) Tyrosine Phosphorylation of the UDP-Glucose Dehydrogenase of Escherichia coli Is at the Crossroads of Colanic Acid Synthesis and Polymyxin Resistance. PLoS ONE 3(8): e3053.
28.Liao CH, Huang YT, Lai CC, Chang CY, Chu FY, Hsu MS, Hsu HS, Hseuh PR.(2011). Klebsiella pneumoniae bacteremia and capsular serotypes, Taiwan. Emerg Infect Dis. 17(6): 1113–1115
29.Lin, M. H., Hsu, T. L., Lin, S. Y., Pan, Y. J., Jan, J. T., Wang, J. T., Khoo, K. H.,and Wu, S. H. (2009). Phosphoproteomics of Klebsiella pneumoniae NTUH- K2044 reveals a tight link between tyrosine phosphorylation and virulence Mol. Cell. Proteomics 8, 2613–2623.
30.Manavalan, P., Johnson, W.C., Jr.(1987). Variable selection method improves the prediction of protein secondary struc- ture from circular dichroism spectra. Anal. Biochem. 167: 76-85, 1987.
31.Manai, M., and Cozzone, A. (1983) Characterization of the amino acids phosphorylated in E. coli proteins. FEMS Microbiol Lett 17: 87-91.
32.Macek, B., Mijakovic, I., Olsen, J.V., Gnad, F., Kumar, C., Jensen, P.R., and Mann, M. (2007) The serine/threonine/tyrosine phosphoproteome of the model bacterium Bacillus subtilis. Mol Cell Proteomics 4: 697–707.
33.Mijakovic, I., Poncet, S., Boel, G., Maze, A., Gillet, S., Jamet, E., Decottignies, P., Grangeasse, C., Doublet, P., Le Marechal, P., and Deutscher, J. (2003) Transmembrane modulator-dependent bacterial tyrosine kinase activates UDP-glucose dehydrogenases. Embo J 22: 4709-4718.
34.Naught, L. E., Regni, C., Beamer, L. J., and Tipton, P. A. (2003) Roles of active site residues in Pseudomonas aeruginosa phosphomannomutase/ phosphoglucomutase, Biochemistry 42: 9946-9951.
35.O’Toole, G.A., and Kolter, R. (1998) The initiation of biofilm formation in Pseudomonas fluorescens WCS365 proceeds via multiple, convergent signaling pathways: a genetic analysis. Mol Microbiol 28: 449–461.
36.Pan PC, Chen HW, Wu PK, Wu YY, Lin CH, Wu JH.(2011) Mutation in fucose synthesis gene of Klebsiella pneumoniae affects capsule composition and virulence in mice. Experimental Biology and Medicine 236: 219–226.
37.Pan, Y. J., Fang, H. C., Yang, H. C., Lin, T. L., Hsieh, P. F., Tsai, F. C., Keynan, Y. & Wang, J. T. (2008). Capsular polysaccharide synthesis regions in Klebsiella pneumoniae serotype K57 and a new capsular serotype. J Clin Microbiol; 46: 2231–2240.
38.Patel G, Huprikar S, Factor SH, Jenkins SG, Calfee DP. (2008) Outcomes of carbapenem-resistant Klebsiella pneumoniae infection and the impact of antimicrobial and adjunctive therapies. Infect Control Hosp Epidemiol ; 29:1099–1106.
39.Peng, H. L., Fu, T. F., Liu, S. F., and Chang, H. Y. (1992) Cloning and Expression of the Klebsiella pneumoniae Galactose Operon. J. Biochem. 112:
604-608
40.Podschun, R., and Ullmann, U. (1998) Klebsiella spp. as nosocomial pathogens: epidemiology, taxonomy, typing methods, and pathogenicity factors. Clin Microbiol Rev 11: 589-603.
41.Rahn, A., Drummelsmith, J., and Whitfield, C. (1999) Conserved organization in the cps gene clusters for expression 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.
42.Ray, W. J.; Post, C. B.; Liu, Y.; Rhyu, G. I. (1993) Structural changes at the metal ion binding site during the phosphoglucomutase reaction. Biochemistry 32,48–57.
43.Regni, C., Schramm, A. M., and Beamer, L. J. (2006) The Reaction of Phosphohexomutase from Pseudomonas aeruginosa: StructuralInsights into a Simple Processive Enzyme. J. Biol. Chem. 281, 15564–15571.
44.Sa-Correia, I., Darzins, A., Wang, S.K., Berry, A., and Chakrabarty, A.M. (1987) Alginate biosynthetic enzymes in mucoid and nonmucoid Pseudomonas aeruginosa: over-production of phosphomannose isomerase, phosphomannomutase, and GDP-mannose pyrophosphorylase by overexpression of the phosphomannose isomerase (pmi) gene. J Bacteriol 169: 3224–3231.
45.Schembri, M.A., Dalsgaard, D., and Klemm, P. (2004) Capsule shields the function of short bacterial adhesins. J Bacteriol 186: 1249–1257.
46.Skorupski, K., and Taylor, R.K.(1996) Positive selection vectors for allelic exchange.Gene 169: 47–52.
47.Simon, R, U. Priefer, and A. Puihler. (1983) A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in gram-negative bacteria. Bio/Iechnology 1:784-791.
48.Soulat D, Jault JM, Duclos B, Geourjon C, Cozzone AJ, Grangeasse C (2006) Staphylococcus aureus operates protein–tyrosine phosphorylation through a specific mechanism. J Biol Chem 281:14048–14056.
49.Van Tilbeurgh H, Declerck N (2001) Structural insights into the regulation of bacterial signalling proteins containing PRDs. Curr Opin Struct Biol 11:685–693
50.Vincent, C., P. Doublet, C. Grangeasse, E. Vaganay, A. J. Cozzone, and B.Duclos. (1999). Cells of Escherichia coli contain a protein-tyrosine kinase,Wzc, and a phosphotyrosine-protein phosphatase, Wzb. J. Bacteriol. 181:3472–3477
51.Wacharotayankun R, Arakawa Y, Ohta M, Hasegawa T, Mori M, Horii T, Kato N. (1992). Involvement of rcsB in Klebsiella K2 capsule synthesis in Escherichia coli K-12. J Bacteriol. 174(3): 1063-7.
52.Whitfield, C., and Roberts, I.S. (1999) Structure, assembly and regulation of expression of capsules in Escherichia coli. Mol Microbiol 31: 1307±1319.
53.Whitfield, C. (2006). Biosynthesis and Assembly of Capsular Polysaccharides in Escherichia coli. Annu. Rev. Biochem. 75:39–68.
54.Wugeditsch T, Paiment A, Hocking J, Drummelsmith J, Forrester C,
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.

連結至畢業學校之論文網頁點我開啟連結
註: 此連結為研究生畢業學校所提供,不一定有電子全文可供下載,若連結有誤,請點選上方之〝勘誤回報〞功能,我們會盡快修正,謝謝!
QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top
無相關期刊