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研究生:黃佳慧
研究生(外文):Chia Hui Huang
論文名稱:探討化膿性鏈球菌 CovS 感知環境中酸性訊號之能力與角色
論文名稱(外文):Role of CovS in sensing acidic signal in Streptococcus pyogenes
指導教授:江倪全
指導教授(外文):C. Chiang-Ni
學位類別:碩士
校院名稱:長庚大學
系所名稱:生物醫學研究所
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:2014
畢業學年度:102
論文頁數:73
中文關鍵詞:化膿性鏈球菌侵襲性感染雙調控系統酸性訊號環境壓力
外文關鍵詞:Streptococcus pyogenesinvasive diseasetwo-component regulatory systemCovRSacidic stress
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誌謝 iii
摘要 iv
Abstract v
目錄 vi
圖目錄 x
表目錄 xi
第一章 緒論 1
1.1. 化膿性鏈球菌之簡介 1
1.2. 化膿性鏈球菌之臨床重要性 1
1.3. 化膿性鏈球菌之毒力因子 2
1.4. 雙調控系統 (Two-component regulatory system) 6
1.5. CovR/CovS (CovRS) 6
1.6. CovRS 突變與侵襲性化膿性鏈球菌感染疾病 8
1.7. 研究目的 10
第二章 材料及方法 11
2.1. 菌株來源 11
2.2. 實驗藥品及溶液配方 11
2.3. 實驗菌株培養及保存 11
2.4. DNA實驗操作 12
2.4.1. 大腸桿菌 (E. coli) 12
2.4.1.1. 質體 DNA 之萃取 12
2.4.1.2. 限制酶酵素切割及 DNA 接合反應 13
2.4.1.3. 勝任細胞 (Competent cell) 之製備 13
2.4.1.4. 大腸桿菌轉型作用 (Transformation) 13
2.4.2. 化膿性鏈球菌 (Streptococcus pyogenes) 14
2.4.2.1. 質體 DNA 之萃取 14
2.4.2.2. 染色體 DNA 之萃取 14
2.4.2.3. 化膿性鏈球菌電穿孔轉型作用 (Electroporation) 15
2.4.3. 聚合酶連鎖反應 (Polymerase chain reaction, PCR) 15
2.5. RNA 實驗操作 16
2.5.1. 化膿性鏈球菌 RNA 之萃取 16
2.5.2. 反轉錄聚合酶連鎖反應 (Reverse transcription PCR, RT-PCR) 16
2.5.3. 即時聚合酶連鎖反應 (Real-time PCR) 17
2.5.4. Promoter activity assay 17
2.5.5. RNA stability assay 18
2.6. 蛋白質實驗操作 18
2.6.1. 蛋白質濃度之測定 18
2.6.2. 蛋白質膠體電泳 (Phos-tag PAGE) 18
2.6.3. 西方點墨法 (Western blotting) 19
2.7. 化膿性鏈球菌表現型之分析 20
2.7.1. 生長曲線之測定 (Growth curve) 20
2.7.2. Skim-milk agar assay 20
2.7.3. 攪拌式發酵槽 20
2.8. 統計方法 21
第三章 結果 22
3.1. AP3 序列之分析 22
3.2. 化膿性鏈球菌在環境壓力下生長之分析 22
3.3. CovR/S 感知酸性訊號能力之分析 23
3.4. covS 互補株之構築與分析 24
3.5. hasA 啟動子活性之分析 24
3.6. hasA RNA stability 之分析 25
3.7. cat RNA stability 之分析 26
3.8. 報導基因 cat 偵測啟動子活性之靈敏性分析 26
3.9. 酸性環境下 covS 自然突變株之篩選 26
3.10. covR 表現量之分析 27
3.11. CovR 磷酸化程度之分析 28
第四章 討論 29
4.1. CovRS 於不同 M 分型菌株對抵禦環境酸鹼值改變之角色 29
4.2. M1T1 A20 化膿性鏈球菌之 CovS 具有感知環境酸性訊號,並抑制目標基因表現之功能 30
4.3. sagA 之表現於 CovS 缺失之菌株中亦受到抑制 30
4.4 報導基因 cat系統不適用於偵測啟動子之活性 31
4.5. CovRS 對目標基因之抑制情形並非因 CovR 之 RNA 或蛋白質表現量不同所造成 32
第五章 總結 33
圖表 34
參考文獻 49
附錄 I 藥品與廠商 58
附錄 II 溶液配方 60

圖目錄
圖一、covS 突變株 AP3 與野生株 A20 部分 covS 基因序列比對結果 38
圖二、A20 和 AP3 於 pH 6.0 和 pH 7.5 TSBY 下之生長曲線 39
圖三、A20、AP3、SCN101、SCN102 在 pH 6.0和pH 7.5 下 hasA、ska 及 sagA 之表現 40
圖四、A20、AP3及 SCN121之 speB 表現 41
圖五、AP3 及 SCN121 在 pH 6.0 和 pH 7.5 下hasA 及 ska 之表現 42
圖六、A20 和 AP3 在 pH 6.0 和 pH 7.5 下 hasA 啟動子之活性 43
圖七、A20 和 AP3 在 pH 6.0 下 hasA RNA 之穩定性 44
圖八、報導基因 cat在 pH 6.0 和 pH 7.5下之 RNA 穩定性 45
圖九、報導基因 cat 偵測啟動子活性與其所測定目標基因之RNA 表現之相關性 46
圖十、A20 及 AP3 在 pH6.0 和 pH 7.5 下 covR 之表現 47
圖十一、A20 及 AP3 在 pH6.0 和 pH 7.5 下 CovR 之磷酸化 48

表目錄
表格一、本論文使用之質體 34
表格二、本論文使用之菌株 35
表格三、本論文使用之引子 36
表格四、 covS 突變株 AP3 與野生株 A20 序列比對結果 37

Ashbaugh CD, Warren HB, Carey VJ &; Wessels MR (1998) Molecular analysis of the role of the group A streptococcal cysteine protease, hyaluronic acid capsule, and M protein in a murine model of human invasive soft-tissue infection. J Clin Invest 102: 550-560.
Ato M, Ikebe T, Kawabata H, Takemori T &; Watanabe H (2008) Incompetence of neutrophils to invasive group A Streptococcus is attributed to induction of plural virulence factors by dysfunction of a regulator. PLoS One 3: e3455.
Aziz RK, Ismail SA, Park HW &; Kotb M (2004) Post-proteomic identification of a novel phage-encoded streptodornase, Sda1, in invasive M1T1 Streptococcus pyogenes. Mol Microbiol 54: 184-197.
Buchanan JT, Simpson AJ, Aziz RK, et al. (2006) DNase expression allows the pathogen group A Streptococcus to escape killing in neutrophil extracellular traps. Curr Biol 16: 396-400.
Carapetis JR, Steer AC, Mulholland EK &; Weber M (2005) The global burden of group A streptococcal diseases. Lancet Infect Dis 5: 685-694.
Chen Z, Itzek A, Malke H, Ferretti JJ &; Kreth J (2012) Dynamics of speB mRNA transcripts in Streptococcus pyogenes. J Bacteriol 194: 1417-1426.
Churchward G, Bates C, Gusa AA, Stringer V &; Scott JR (2009) Regulation of streptokinase expression by CovR/S in Streptococcus pyogenes: CovR acts through a single high-affinity binding site. Microbiology 155: 566-575.
Cleary PP, Prahbu U, Dale JB, Wexler DE &; Handley J (1992) Streptococcal C5a peptidase is a highly specific endopeptidase. Infect Immun 60: 5219-5223.
Cole JN, Barnett TC, Nizet V &; Walker MJ (2011) Molecular insight into invasive group A streptococcal disease. Nature Rev Microbiol 9: 724-736.
Cunningham MW (2000) Pathogenesis of group A streptococcal infections. Clin Microbiol Rev 13: 470-511.
Cywes C &; Wessels MR (2001) Group A Streptococcus tissue invasion by CD44-mediated cell signalling. Nature 414: 648-652.
Dale JB, Washburn RG, Marques MB &; Wessels MR (1996) Hyaluronate capsule and surface M protein in resistance to opsonization of group A streptococci. Infect Immun 64: 1495-1501.
Dalton TL &; Scott JR (2004) CovS inactivates CovR and is required for growth under conditions of general stress in Streptococcus pyogenes. J Bacteriol 186: 3928-3937.
Datta V, Myskowski SM, Kwinn LA, et al. (2005) Mutational analysis of the group A streptococcal operon encoding streptolysin S and its virulence role in invasive infection. Mol Microbiol 56: 681-695.
Engleberg NC, Heath A, Miller A, Rivera C &; DiRita VJ (2001) Spontaneous mutations in the CsrRS two-component regulatory system of Streptococcus pyogenes result in enhanced virulence in a murine model of skin and soft tissue infection. J Infect Dis 183: 1043-1054.
Facklam R, Beall B, Efstratiou A, et al. (1999) emm typing and validation of provisional M types for group A streptococci. Emerg Infect Dis 5: 247-253.
Federle MJ &; Scott JR (2002) Identification of binding sites for the group A streptococcal global regulator CovR. Mol Microbiol 43: 1161-1172.
Gao J, Gusa AA, Scott JR &; Churchward G (2005) Binding of the global response regulator protein CovR to the sag promoter of Streptococcus pyogenes reveals a new mode of CovR-DNA interaction. J Biol Chem 280: 38948-38956.
Graham MR, Smoot LM, Migliaccio CA, et al. (2002) Virulence control in group A Streptococcus by a two-component gene regulatory system: global expression profiling and in vivo infection modeling. Proc Natl Acad Sci USA 99: 13855-13860.
Gryllos I, Levin JC &; Wessels MR (2003) The CsrR/CsrS two-component system of group A Streptococcus responds to environmental Mg2+. Proc Natl Acad Sci USA 100: 4227-4232.
Ikebe T, Ato M, Matsumura T, Hasegawa H, Sata T, Kobayashi K &; Watanabe H (2010) Highly frequent mutations in negative regulators of multiple virulence genes in group A streptococcal toxic shock syndrome isolates. PLoS Pathog 6: e1000832.
Ji Y, McLandsborough L, Kondagunta A &; Cleary PP (1996) C5a peptidase alters clearance and trafficking of group A streptococci by infected mice. Infect Immun 64: 503-510.
Ji YD, Carlson B, Kondagunta A &; Cleary PP (1997) Intranasal immunization with C5a peptidase prevents nasopharyngeal colonization of mice by the group A Streptococcus. Infection and Immunity 65: 2080-2087.
Kamezawa Y, Nakahara T, Nakano S, Abe Y, Nozaki-Renard J &; Isono T (1997) Streptococcal mitogenic exotoxin Z, a novel acidic superantigenic toxin produced by a T1 strain of Streptococcus pyogenes. Infect Immun 65: 3828-3833.
Khil J, Im M, Heath A, Ringdahl U, Mundada L, Cary Engleberg N &; Fay WP (2003) Plasminogen enhances virulence of group A streptococci by streptokinase-dependent and streptokinase-independent mechanisms. J Infect Dis 188: 497-505.
Kobayashi SD, Braughton KR, Whitney AR, Voyich JM, Schwan TG, Musser JM &; DeLeo FR (2003) Bacterial pathogens modulate an apoptosis differentiation program in human neutrophils. Proc Natl Acad Sci USA 100: 10948-10953.
Kreikemeyer B, McIver KS &; Podbielski A (2003) Virulence factor regulation and regulatory networks in Streptococcus pyogenes and their impact on pathogen–host interactions. Trends Microbiol 11: 224-232.
Lancefield RC (1993) A serological differentiation of human and other groups of hemolytic streptococci. J Exp Med 57: 571-595.
Li J, Zhu H, Feng W, et al. (2013) Regulation of inhibition of neutrophil infiltration by the two-component regulatory system CovRS in subcutaneous murine infection with group A Streptococcus. Infect Immun 81: 974-983.
Li Z, Ploplis VA, French EL &; Boyle MD (1999) Interaction between group A streptococci and the plasmin(ogen) system promotes virulence in a mouse skin infection model. J Infect Dis 179: 907-914.
Limbago B, Penumalli V, Weinrick B &; Scott JR (2000) Role of streptolysin O in a mouse model of invasive group A streptococcal disease. Infect Immun 68: 6384-6390.
Luk EY, Lo JY, Li AZ, et al. (2012) Scarlet fever epidemic, Hong Kong, 2011. Emerg Infect Dis 18: 1658-1661.
Mascher T, Helmann JD &; Unden G (2006) Stimulus perception in bacterial signal-transducing histidine kinases. Microbiol Mol Biol Rev 70: 910-938.
Metzgar D &; Zampolli A (2011) The M protein of group A Streptococcus is a key virulence factor and a clinically relevant strain identification marker. Virulence 2: 402-412.
Mitrophanov AY, Churchward G &; Borodovsky M (2007) Control of Streptococcus pyogenes virulence: modeling of the CovR/S signal transduction system. J Theor Biol 246: 113-128.
Moses AE, Wessels MR, Zalcman K, Alberti S, Natanson-Yaron S, Menes T &; Hanski E (1997) Relative contributions of hyaluronic acid capsule and M protein to virulence in a mucoid strain of the group A Streptococcus. Infect Immun 65: 64-71.
Ohara-Nemoto Y, Sasaki M, Kaneko M, Nemoto T &; Ota M (1994) Cysteine protease activity of streptococcal pyrogenic exotoxin B. Can J Microbiol 40: 930-936.
Olsen RJ &; Musser JM (2010) Molecular pathogenesis of necrotizing fasciitis. Annu Rev Pathol 5: 1-31.
Raeder R, Harokopakis E, Hollingshead S &; Boyle MD (2000) Absence of SpeB production in virulent large capsular forms of group A streptococcal strain 64. Infect Immun 68: 744-751.
Ravins M, Jaffe J, Hanski E, Shetzigovski I, Natanson-Yaron S &; Moses AE (2000) Characterization of a mouse-passaged, highly encapsulated variant of group A Streptococcus in in vitro and in vivo studies. J Infect Dis 182: 1702-1711.
Ribardo DA, Lambert TJ &; McIver KS (2004) Role of Streptococcus pyogenes two-component response regulators in the temporal control of Mga and the Mga-regulated virulence gene emm. Infect Immun 72: 3668-3673.
Sandin C, Carlsson F &; Lindahl G (2006) Binding of human plasma proteins to Streptococcus pyogenes M protein determines the location of opsonic and non-opsonic epitopes. Mol Microbiol 59: 20-30.
Savic DJ &; McShan WM (2012) Long-term survival of Streptococcus pyogenes in rich media is pH-dependent. Microbiology 158: 1428-1436.
Skerker JM, Prasol MS, Perchuk BS, Biondi EG &; Laub MT (2005) Two-component signal transduction pathways regulating growth and cell cycle progression in a bacterium: a system-level analysis. PLoS Biol 3: e334.
Smeesters PR, McMillan DJ &; Sriprakash KS (2010) The streptococcal M protein: a highly versatile molecule. Trends Microbiol 18: 275-282.
Staali L, Bauer S, Morgelin M, Bjorck L &; Tapper H (2006) Streptococcus pyogenes bacteria modulate membrane traffic in human neutrophils and selectively inhibit azurophilic granule fusion with phagosomes. Cell Microbiol 8: 690-703.
Steer AC, Batzloff MR, Mulholland K &; Carapetis JR (2009) Group A streptococcal vaccines: facts versus fantasy. Curr Opin Infect Dis 22: 544-552.
Stock AM, Robinson VL &; Goudreau PN (2000) Two-component signal transduction. Annu Rev Biochem 69: 183-215.
Sumby P, Whitney AR, Graviss EA, DeLeo FR &; Musser JM (2006) Genome-wide analysis of group a streptococci reveals a mutation that modulates global phenotype and disease specificity. PLoS Pathog 2: e5.
Sumby P, Barbian KD, Gardner DJ, et al. (2005) Extracellular deoxyribonuclease made by group A Streptococcus assists pathogenesis by enhancing evasion of the innate immune response. Proc Natl Acad Sci USA 102: 1679-1684.
Sumitomo T, Nakata M, Higashino M, Jin Y, Terao Y, Fujinaga Y &; Kawabata S (2011) Streptolysin S contributes to group A streptococcal translocation across an epithelial barrier. J Biol Chem 286: 2750-2761.
Tamayo E, Montes M, Garcia-Medina G, Garcia-Arenzana JM &; Perez-Trallero E (2010) Spread of a highly mucoid Streptococcus pyogenes emm3/ST15 clone. BMC Infect Dis 10: 233-237.
Tatsuno I, Okada R, Zhang Y, Isaka M &; Hasegawa T (2013) Partial loss of CovS function in Streptococcus pyogenes causes severe invasive disease. BMC Res Notes 6: 126-134.
Toyosaki T, Yoshioka T, Tsuruta Y, Yutsudo T, Iwasaki M &; Suzuki R (1996) Definition of the mitogenic factor (MF) as a novel streptococcal superantigen that is different from streptococcal pyrogenic exotoxins A, B, and C. Eur J Immunol 26: 2693-2701.
Tran-Winkler HJ, Love JF, Gryllos I &; Wessels MR (2011) Signal transduction through CsrRS confers an invasive phenotype in group A Streptococcus. PLoS Pathog 7: e1002361.
Trevino J, Perez N, Ramirez-Pena E, Liu Z, Shelburne SA, Musser JM &; Sumby P (2009) CovS simultaneously activates and inhibits the CovR-mediated repression of distinct subsets of group A Streptococcus virulence factor-encoding genes. Infect Immun 77: 3141-3149.
Walker MJ, Hollands A, Sanderson-Smith ML, et al. (2007) DNase Sda1 provides selection pressure for a switch to invasive group A streptococcal infection. Nat Med 13: 981-985.

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