(3.230.76.48) 您好!臺灣時間:2021/04/11 09:35
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:::

我願授權國圖
: 
twitterline
研究生:賴思羽
研究生(外文):Szu-Yu Lai
論文名稱:探討small RNA Spot42在奇異變形桿菌中所扮演的角色
論文名稱(外文):Investigation of the small RNA Spot42 in uropathogenic Proteus mirabilis
指導教授:廖淑貞廖淑貞引用關係
口試委員:鄧麗珍楊翠青
口試日期:2016-07-25
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:醫學檢驗暨生物技術學研究所
學門:醫藥衛生學門
學類:醫學技術及檢驗學類
論文種類:學術論文
論文出版年:2016
畢業學年度:104
語文別:中文
論文頁數:102
中文關鍵詞:奇異變形桿菌Spot42
外文關鍵詞:Proteus mirabilissmall RNASpot42
相關次數:
  • 被引用被引用:0
  • 點閱點閱:179
  • 評分評分:系統版面圖檔系統版面圖檔系統版面圖檔系統版面圖檔系統版面圖檔
  • 下載下載:0
  • 收藏至我的研究室書目清單書目收藏:0
  奇異變形桿菌 (Proteus mirabilis)是革蘭氏陰性的兼性厭氧菌,於健康人體腸道內屬正常菌叢,然而在腸道以外的地方則會造成伺機性感染 (opportunistic infection),主要會造成長期插導尿管或泌尿道功能缺失的病人之泌尿道感染(urinary tract infection, UTI)。細菌發展出許多調控機制,使其能夠適應週遭多樣且波動的環境。目前在Escherichia coli已被發現有許多sRNA會參與調控細菌的壓力反應,其中包含sRNA Spot42 (或稱spf)。在E. coli中發現當環境中葡萄糖濃度增加時Spot42會大量表現,抑制galK (encodes a galactokinase)轉譯,此外也參與在調控至少22個和次級碳源的攝取與代謝相關基因表現。在壓力調控方面則是發現Spot42會正向調控general stress response regulator, RpoS的mRNA表現,使細菌增加抵抗酸性壓力的能力,但是否會影響細菌適應其他不利的環境及調控機制仍不甚了解。本研究旨在探討Spot42在P. mirabilis中所扮演的角色。我們建構spf突變株,在表現型方面發現spf突變並不影響細菌生長,此外也發現spf突變會降低細菌的抗酸及抗氧化壓力的能力、在巨噬細胞 (THP-1)的存活率及對膀胱細胞 (NTUB1)及腎臟細胞 (A498)的入侵及貼附能力。而利用pGEM-T easy載體在野生株中過度表現spf則發現不會影響細菌移動性、抗藥性、和生物膜的形成。在基因調控方面,先前的研究指出CRP-cAMP complex會結合到spf的啟動子區域而抑制其轉錄並且負向調控rpoS mRNA表現,我們分別利用EMSA及realtime PCR證實CRP-cAMP complex會結合至spf的啟動子區域並負向調控其表現;利用reporter assay發現CRP會促進rpoS轉錄但realtime PCR的結果則顯示CRP會負向調控rpoS mRNA表現;分析Spot42對rpoS的調控發現Spot42會正向調控rpoS mRNA的表現,我們進一步利用生物資訊網站預測也在 rpoS發現Spot42 binding site。綜合上述結果我們認為在P. mirabilis中Spot42受到CRP的直接調控,進而影響rpoS表現造成壓力抵抗相關的表現型。

Proteus mirabilis, a Gram-negative, facultative anaerobic bacteria, is one of the most common cause of urinary tract infections, especially in patients with indwelling catheters or structural abnormalities of the urinary tract. The bacteria have developed diverse response systems to survive in the harsh and changing conditions. Many Hfq-binding small RNAs have been found to play roles in stress responses in E. coli and S. typhimurium. The Spot42 RNA, a 109 nucleotides long, Hfq-dependent small non-coding RNA, has been reported to block the translation of galK gene in E. coli under growth in the presence of glucose and regulate over twenty-two genes associated with uptake and catabolism of non-favored carbon sources. On the other hand, Spot42 was also found to participate in bacterial acid resistance and upregulate the general stress response regulator, RpoS. But the connection between Spot42 and the bacterial stress responses is still poorly understood. In this study, we constructed spf deletion mutant to investigate the effect of Proteus mirabilis Spot42 on adaption to stress responses and the regulatory mechanisms. We found that spf mutation decreaced acidic (pH=3) and oxidative stress resistance (30 mM H2O2), the survival in macrophage, and the ability to invade and adhere to NTUB1 and A498 cells compared with wild-type strain. In addition, we also noticed that spf has nothing to do with cell growth, swarming motility, biofilm formation and the sensitivity to antibiotics. In the aspect of gene regulation, we confirmed that CRP-cAMP binds to spf promoter region and negatively controls the expression of Spot42 in P. mirabilis by EMSA and realtime PCR respectively. Further, the realtime PCR data indicates that Spot42 would positively regulate the expression of rpoS mRNA level. We used bioinformatic tools to predict RNA-RNA interactions, and also found out the binding site of Spot42 on rpoS mRNA. The realtime PCR data also demonstrates that CRP negatively regulates the expression of rpoS mRNA level. In summury, our data suggest that Spot42 is important in P. mirabilis stress resistance by either directly or indirectly modulating the expression of RpoS.

誌謝 i
摘要 ii
Abstract iii
目錄 iv
表目錄 v
圖目錄 vi
第一章 緒論 1
第一節 奇異變形桿菌(Proteus mirabilis)介紹 1
第二節 CRP、Spot42 sRNA及RpoS的基本介紹 7
第三節 研究動機與目的 11
第四節 實驗設計 12
第二章 實驗材料與方法 13
第一節 實驗材料 13
第二節 Spot42突變株建構方法 16
第三節 分析表現型 (phenotype)及毒力因子 (virulence factor) 32
第四節 Spot42參與之基因調控 47
第三章 結果 56
第一節 P. mirabilis spf突變株之建立與確認 56
第二節 spf突變菌株之表現型分析 57
第三節 spf突變株毒力因子之分析 59
第四節 分析spf可能調控的路徑 60
第四章 結論與討論 63
第一節 結論 63
第二節 討論 64
第三節 未來展望 67
第五章 表 68
第六章 圖 72
附錄 87
參考文獻 96



1.Jacobsen, S.M. and M.E. Shirtliff, Proteus mirabilis biofilms and catheter-associated urinary tract infections. Virulence, 2011. 2(5): p. 460-5.
2.Warren, J.W., et al., A prospective microbiologic study of bacteriuria in patients with chronic indwelling urethral catheters. J Infect Dis, 1982. 146(6): p. 719-23.
3.Hooton, T.M., et al., Diagnosis, prevention, and treatment of catheter-associated urinary tract infection in adults: 2009 International Clinical Practice Guidelines from the Infectious Diseases Society of America. Clin Infect Dis, 2010. 50(5): p. 625-63.
4.Jacobsen, S.M., et al., Complicated catheter-associated urinary tract infections due to Escherichia coli and Proteus mirabilis. Clin Microbiol Rev, 2008. 21(1): p. 26-59.
5.Armbruster, C.E. and H.L. Mobley, Merging mythology and morphology: the multifaceted lifestyle of Proteus mirabilis. Nat Rev Microbiol, 2012. 10(11): p. 743-54.
6.Mobley, H.L., et al., Construction of a flagellum-negative mutant of Proteus mirabilis: effect on internalization by human renal epithelial cells and virulence in a mouse model of ascending urinary tract infection. Infect Immun, 1996. 64(12): p. 5332-40.
7.Allison, C., et al., The role of swarm cell differentiation and multicellular migration in the uropathogenicity of Proteus mirabilis. J Infect Dis, 1994. 169(5): p. 1155-8.
8.Belas, R., D. Erskine, and D. Flaherty, Proteus mirabilis mutants defective in swarmer cell differentiation and multicellular behavior. J Bacteriol, 1991. 173(19): p. 6279-88.
9.Pearson, M.M., et al., Complete genome sequence of uropathogenic Proteus mirabilis, a master of both adherence and motility. J Bacteriol, 2008. 190(11): p. 4027-37.
10.Baldo, C. and S.P.D. Rocha, Virulence Factors Of Uropathogenic Proteus Mirabilis-A Mini Review.
11.Rocha, S.P., J.S. Pelayo, and W.P. Elias, Fimbriae of uropathogenic Proteus mirabilis. FEMS Immunol Med Microbiol, 2007. 51(1): p. 1-7.
12.Li, X., D.E. Johnson, and H.L. Mobley, Requirement of MrpH for mannose-resistant Proteus-like fimbria-mediated hemagglutination by Proteus mirabilis. Infect Immun, 1999. 67(6): p. 2822-33.
13.Silverblatt, F.J. and I. Ofek, Influence of pili on the virulence of Proteus mirabilis in experimental hematogenous pyelonephritis. J Infect Dis, 1978. 138(5): p. 664-7.
14.Pellegrino, R., et al., Proteus mirabilis uroepithelial cell adhesin (UCA) fimbria plays a role in the colonization of the urinary tract. Pathog Dis, 2013. 67(2): p. 104-7.
15.Bahrani, F.K., et al., Construction of an MR/P fimbrial mutant of Proteus mirabilis: role in virulence in a mouse model of ascending urinary tract infection. Infect Immun, 1994. 62(8): p. 3363-71.
16.Flemming, H.C. and J. Wingender, The biofilm matrix. Nat Rev Microbiol, 2010. 8(9): p. 623-33.
17.McDougald, D., et al., Should we stay or should we go: mechanisms and ecological consequences for biofilm dispersal. Nat Rev Microbiol, 2012. 10(1): p. 39-50.
18.Mobley, H.L., M.D. Island, and R.P. Hausinger, Molecular biology of microbial ureases. Microbiol Rev, 1995. 59(3): p. 451-80.
19.Griffith, D.P., D.M. Musher, and C. Itin, Urease. The primary cause of infection-induced urinary stones. Invest Urol, 1976. 13(5): p. 346-50.
20.Li, X., et al., Visualization of Proteus mirabilis within the matrix of urease-induced bladder stones during experimental urinary tract infection. Infect Immun, 2002. 70(1): p. 389-94.
21.Drechsel, H., et al., Alpha-keto acids are novel siderophores in the genera Proteus, Providencia, and Morganella and are produced by amino acid deaminases. J Bacteriol, 1993. 175(9): p. 2727-33.
22.Walker, K.E., et al., ZapA, the IgA-degrading metalloprotease of Proteus mirabilis, is a virulence factor expressed specifically in swarmer cells. Mol Microbiol, 1999. 32(4): p. 825-36.
23.Braun, V. and T. Focareta, Pore-forming bacterial protein hemolysins (cytolysins). Crit Rev Microbiol, 1991. 18(2): p. 115-58.
24.Mobley, H.L., et al., Cytotoxicity of the HpmA hemolysin and urease of Proteus mirabilis and Proteus vulgaris against cultured human renal proximal tubular epithelial cells. Infect Immun, 1991. 59(6): p. 2036-42.
25.Allison, C. and C. Hughes, Bacterial swarming: an example of prokaryotic differentiation and multicellular behaviour. Sci Prog, 1991. 75(298 Pt 3-4): p. 403-22.
26.Coker, C., et al., Pathogenesis of Proteus mirabilis urinary tract infection. Microbes Infect, 2000. 2(12): p. 1497-505.
27.Rauprich, O., et al., Periodic phenomena in Proteus mirabilis swarm colony development. J Bacteriol, 1996. 178(22): p. 6525-38.
28.Givskov, M., et al., Two separate regulatory systems participate in control of swarming motility of Serratia liquefaciens MG1. J Bacteriol, 1998. 180(3): p. 742-5.
29.Leon, R. and G. Espin, flhDC, but not fleQ, regulates flagella biogenesis in Azotobacter vinelandii, and is under AlgU and CydR negative control. Microbiology, 2008. 154(Pt 6): p. 1719-28.
30.Stickler, D., et al., Studies on the formation of crystalline bacterial biofilms on urethral catheters. Eur J Clin Microbiol Infect Dis, 1998. 17(9): p. 649-52.
31.Fraser, G.M., et al., Swarming-coupled expression of the Proteus mirabilis hpmBA haemolysin operon. Microbiology, 2002. 148(Pt 7): p. 2191-201.
32.Allison, C., et al., Ability of Proteus mirabilis to invade human urothelial cells is coupled to motility and swarming differentiation. Infect Immun, 1992. 60(11): p. 4740-6.
33.Hay, N.A., et al., A nonswarming mutant of Proteus mirabilis lacks the Lrp global transcriptional regulator. J Bacteriol, 1997. 179(15): p. 4741-6.
34.Nielubowicz, G.R., S.N. Smith, and H.L. Mobley, Outer membrane antigens of the uropathogen Proteus mirabilis recognized by the humoral response during experimental murine urinary tract infection. Infect Immun, 2008. 76(9): p. 4222-31.
35.Ragnarsdottir, B., et al., Genetics of innate immunity and UTI susceptibility. Nat Rev Urol, 2011. 8(8): p. 449-68.
36.Weichhart, T., et al., Current concepts of molecular defence mechanisms operative during urinary tract infection. Eur J Clin Invest, 2008. 38 Suppl 2: p. 29-38.
37.Drake, R., Genetics: TLR4 promoter variants influence response to UTI. Nat Rev Urol, 2010. 7(7): p. 365.
38.Pearson, M.M., et al., Transcriptome of Proteus mirabilis in the murine urinary tract: virulence and nitrogen assimilation gene expression. Infect Immun, 2011. 79(7): p. 2619-31.
39.Zheng, D., et al., Identification of the CRP regulon using in vitro and in vivo transcriptional profiling. Nucleic Acids Res, 2004. 32(19): p. 5874-93.
40.Grainger, D.C., et al., Studies of the distribution of Escherichia coli cAMP-receptor protein and RNA polymerase along the E. coli chromosome. Proc Natl Acad Sci U S A, 2005. 102(49): p. 17693-8.
41.Gorke, B. and J. Stulke, Carbon catabolite repression in bacteria: many ways to make the most out of nutrients. Nat Rev Microbiol, 2008. 6(8): p. 613-24.
42.Landis, L., J. Xu, and R.C. Johnson, The cAMP receptor protein CRP can function as an osmoregulator of transcription in Escherichia coli. Genes Dev, 1999. 13(23): p. 3081-91.
43.Johansson, J., et al., Nucleoid proteins stimulate stringently controlled bacterial promoters: a link between the cAMP-CRP and the (p)ppGpp regulons in Escherichia coli. Cell, 2000. 102(4): p. 475-85.
44.Castanie-Cornet, M.P. and J.W. Foster, Escherichia coli acid resistance: cAMP receptor protein and a 20 bp cis-acting sequence control pH and stationary phase expression of the gadA and gadBC glutamate decarboxylase genes. Microbiology, 2001. 147(Pt 3): p. 709-15.
45.Uppal, S. and N. Jawali, Cyclic AMP receptor protein (CRP) regulates the expression of cspA, cspB, cspG and cspI, members of cspA family, in Escherichia coli. Arch Microbiol, 2015. 197(3): p. 497-501.
46.Jackson, D.W., J.W. Simecka, and T. Romeo, Catabolite repression of Escherichia coli biofilm formation. J Bacteriol, 2002. 184(12): p. 3406-10.
47.Balsalobre, C., J. Johansson, and B.E. Uhlin, Cyclic AMP-dependent osmoregulation of crp gene expression in Escherichia coli. J Bacteriol, 2006. 188(16): p. 5935-44.
48.Tian, Z.X., et al., The CRP-cAMP complex and downregulation of the glnAp2 promoter provides a novel regulatory linkage between carbon metabolism and nitrogen assimilation in Escherichia coli. Mol Microbiol, 2001. 41(4): p. 911-24.
49.Zhang, Z., et al., Functional interactions between the carbon and iron utilization regulators, Crp and Fur, in Escherichia coli. J Bacteriol, 2005. 187(3): p. 980-90.
50.Nishino, K., Y. Senda, and A. Yamaguchi, CRP regulator modulates multidrug resistance of Escherichia coli by repressing the mdtEF multidrug efflux genes. J Antibiot (Tokyo), 2008. 61(3): p. 120-7.
51.De Lay, N. and S. Gottesman, The Crp-activated small noncoding regulatory RNA CyaR (RyeE) links nutritional status to group behavior. J Bacteriol, 2009. 191(2): p. 461-76.
52.Deutscher, J., The mechanisms of carbon catabolite repression in bacteria. Curr Opin Microbiol, 2008. 11(2): p. 87-93.
53.Hogema, B.M., et al., Inducer exclusion in Escherichia coli by non-PTS substrates: the role of the PEP to pyruvate ratio in determining the phosphorylation state of enzyme IIAGlc. Mol Microbiol, 1998. 30(3): p. 487-98.
54.Hanamura, A. and H. Aiba, Molecular mechanism of negative autoregulation of Escherichia coli crp gene. Nucleic Acids Res, 1991. 19(16): p. 4413-9.
55.Berg, O.G. and P.H. von Hippel, Selection of DNA binding sites by regulatory proteins. II. The binding specificity of cyclic AMP receptor protein to recognition sites. J Mol Biol, 1988. 200(4): p. 709-23.
56.Papenfort, K. and J. Vogel, Sweet business: Spot42 RNA networks with CRP to modulate catabolite repression. Mol Cell, 2011. 41(3): p. 245-6.
57.Wu, J., et al., Pyruvate-associated acid resistance in bacteria. Appl Environ Microbiol, 2014. 80(14): p. 4108-13.
58.Guo, M., et al., Positive Effect of Carbon Sources on Natural Transformation in Escherichia coli: Role of Low-Level Cyclic AMP (cAMP)-cAMP Receptor Protein in the Derepression of rpoS. J Bacteriol, 2015. 197(20): p. 3317-28.
59.Storz, G., J. Vogel, and K.M. Wassarman, Regulation by small RNAs in bacteria: expanding frontiers. Mol Cell, 2011. 43(6): p. 880-91.
60.Murina, V.N. and A.D. Nikulin, Bacterial Small Regulatory RNAs and Hfq Protein. Biochemistry (Mosc), 2015. 80(13): p. 1647-54.
61.Mura, C., et al., Archaeal and eukaryotic homologs of Hfq: A structural and evolutionary perspective on Sm function. RNA Biol, 2013. 10(4): p. 636-51.
62.Wang, M.C., et al., The RNA chaperone Hfq is involved in stress tolerance and virulence in uropathogenic Proteus mirabilis. PLoS One, 2014. 9(1): p. e85626.
63.Joyce, C.M. and N.D. Grindley, Identification of two genes immediately downstream from the polA gene of Escherichia coli. J Bacteriol, 1982. 152(3): p. 1211-9.
64.Polayes, D.A., et al., Cyclic AMP-cyclic AMP receptor protein as a repressor of transcription of the spf gene of Escherichia coli. J Bacteriol, 1988. 170(7): p. 3110-4.
65.Beisel, C.L. and G. Storz, The base-pairing RNA spot 42 participates in a multioutput feedforward loop to help enact catabolite repression in Escherichia coli. Mol Cell, 2011. 41(3): p. 286-97.
66.Moller, T., et al., Spot 42 RNA mediates discoordinate expression of the E. coli galactose operon. Genes Dev, 2002. 16(13): p. 1696-706.
67.Wang, X., et al., Two-level inhibition of galK expression by Spot 42: Degradation of mRNA mK2 and enhanced transcription termination before the galK gene. Proc Natl Acad Sci U S A, 2015. 112(24): p. 7581-6.
68.Beisel, C.L., et al., Multiple factors dictate target selection by Hfq-binding small RNAs. Embo j, 2012. 31(8): p. 1961-74.
69.Tanabe, T., et al., The small RNA Spot 42 regulates the expression of the type III secretion system 1 (T3SS1) chaperone protein VP1682 in Vibrio parahaemolyticus. FEMS Microbiol Lett, 2015. 362(21).
70.Weber, H., et al., Genome-wide analysis of the general stress response network in Escherichia coli: sigmaS-dependent genes, promoters, and sigma factor selectivity. J Bacteriol, 2005. 187(5): p. 1591-603.
71.Battesti, A., N. Majdalani, and S. Gottesman, The RpoS-mediated general stress response in Escherichia coli. Annu Rev Microbiol, 2011. 65: p. 189-213.
72.Aiba, H., Mechanism of RNA silencing by Hfq-binding small RNAs. Curr Opin Microbiol, 2007. 10(2): p. 134-9.
73.Soper, T., et al., Positive regulation by small RNAs and the role of Hfq. Proc Natl Acad Sci U S A, 2010. 107(21): p. 9602-7.
74.Sousa, S.A., et al., The hfq gene is required for stress resistance and full virulence of Burkholderia cepacia to the nematode Caenorhabditis elegans. Microbiology, 2010. 156(Pt 3): p. 896-908.
75.Artee and Kaushala, Microbial Stress Response Regulatory Enzyme and Their Pharmaceutical Application Int. J. Curr. Res. Chem. Pharma. Sci., 2015. 2(8): p. 59-66.
76.Barth, E., et al., Interplay of cellular cAMP levels, {sigma}S activity and oxidative stress resistance in Escherichia coli. Microbiology, 2009. 155(Pt 5): p. 1680-9.
77.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.
78.Wu, Y. and F.W. Outten, IscR controls iron-dependent biofilm formation in Escherichia coli by regulating type I fimbria expression. J Bacteriol, 2009. 191(4): p. 1248-57.
79.Chiang, M.K., et al., Impact of Hfq on global gene expression and virulence in Klebsiella pneumoniae. PLoS One, 2011. 6(7): p. e22248.
80.Fukuoka, T., et al., Increase in susceptibility of Pseudomonas aeruginosa to carbapenem antibiotics in low-amino-acid media. Antimicrob Agents Chemother, 1991. 35(3): p. 529-32.
81.Del Porto, P., et al., Dysfunctional CFTR alters the bactericidal activity of human macrophages against Pseudomonas aeruginosa. PLoS One, 2011. 6(5): p. e19970.
82.Jiang, S.S., et al., Proteus mirabilis pmrI, an RppA-regulated gene necessary for polymyxin B resistance, biofilm formation, and urothelial cell invasion. Antimicrob Agents Chemother, 2010. 54(4): p. 1564-71.
83.Schweizer, H.P. and T.T. Hoang, An improved system for gene replacement and xylE fusion analysis in Pseudomonas aeruginosa. Gene, 1995. 158(1): p. 15-22.
84.Jiang, S.S., et al., Characterization of UDP-glucose dehydrogenase and UDP-glucose pyrophosphorylase mutants of Proteus mirabilis: defectiveness in polymyxin B resistance, swarming, and virulence. Antimicrob Agents Chemother, 2010. 54(5): p. 2000-9.
85.Hatfull, G.F. and C.M. Joyce, Deletion of the spf (spot 42 RNA) gene of Escherichia coli. J Bacteriol, 1986. 166(3): p. 746-50.
86.Gosset, G., et al., Transcriptome analysis of Crp-dependent catabolite control of gene expression in Escherichia coli. J Bacteriol, 2004. 186(11): p. 3516-24.
87.Kulesus, R.R., et al., Impact of the RNA chaperone Hfq on the fitness and virulence potential of uropathogenic Escherichia coli. Infect Immun, 2008. 76(7): p. 3019-26.
88.Geng, J., et al., Involvement of the post-transcriptional regulator Hfq in Yersinia pestis virulence. PLoS One, 2009. 4(7): p. e6213.
89.Hansen, G.A., et al., Expression profiling reveals Spot 42 small RNA as a key regulator in the central metabolism of Aliivibrio salmonicida. BMC Genomics, 2012. 13: p. 37.
90.Schaffer, J.N. and M.M. Pearson, Proteus mirabilis and Urinary Tract Infections. Microbiol Spectr, 2015. 3(5).
91.Wright, P.R., et al., CopraRNA and IntaRNA: predicting small RNA targets, networks and interaction domains. Nucleic Acids Res, 2014. 42(Web Server issue): p. W119-23.
92.Lange, R. and R. Hengge-Aronis, The cellular concentration of the sigma S subunit of RNA polymerase in Escherichia coli is controlled at the levels of transcription, translation, and protein stability. Genes Dev, 1994. 8(13): p. 1600-12.



QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top
系統版面圖檔 系統版面圖檔