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研究生:王志宏
研究生(外文):CHIH-HUNG WANG
論文名稱:寡胜肽膜透酶在A群鏈球菌感染過程中之角色
論文名稱(外文):Roles of Oligopeptide Permease in Group A Streptococcal Infection
指導教授:吳俊忠
指導教授(外文):Jiunn-Jong Wu
學位類別:博士
校院名稱:國立成功大學
系所名稱:基礎醫學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2005
畢業學年度:93
語文別:中文
論文頁數:147
中文關鍵詞:寡胜肽膜透酶A 群鏈球菌
外文關鍵詞:oligopeptide permeaseStreptococcus pyogenes
相關次數:
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  A 群鏈球菌 (Group A Streptococcus, GAS) 為重要的人類病原菌可以造成呼吸道及其他組織的感染。 A 群鏈球菌之寡胜肽膜透酶 (oligopeptide permease, Opp) 為表面蛋白, 屬於 ABC-transporter family 的一支。 opp 為一 polycistronic operon, 由 oppA, oppB, oppC, oppD 和 oppF 5 個基因組合而成。 寡胜肽膜透酶在 A 群鏈球菌的生物功能仍不清楚, 故為釐清寡胜肽膜透酶在 A 群鏈球菌感染過程中所扮演之角色, 本研究利 integrative 質體進行 opp 突變株之構築, 並以南方墨漬法及 RT-PCR 確認。 本研究發現 opp 突變株不僅可減少 speB、speX 和 rofA 的表現外, 亦可增加 speF、 sagA (streptolysin S associated gene A)、 slo (streptolysin O)、 pel (pleotrophic effect locus) 和 dppA (dipeptide permease) 的表現, 但不影響 emm、 speJ、 speG、 rgg 和 csrR 的表現。 此外, 與野生株比較, 對 BALB/c 小鼠而言, opp 突變株具有高散佈能力與低致死率, 且較不易造成組織傷害。 比較 opp 突變株與野生株對細胞的附著與侵入率, 突變株較野生株分別減少約 50% 和 46%。 利用 pull down 和西方墨點法證明在 GAS 感染過程, 寡胜肽膜透酶可引發 A549 細胞之 Rac 和 Rho 的活化以及 FAK 和JNK 的磷酸化以造成細胞骨架重組。 由免疫螢光與電子顯微鏡的結果顯示,A 群鏈球菌可藉由寡胜肽膜透酶打開 A549 與 MDCK 細胞的細胞緊密接合 (tight junction), 而使 A 群鏈球菌可能深入至較深層組織。 根據以上的結果, 本研究推論 opp operon 在 A 群鏈球菌感染過程中扮演兩個重要的角色。 首先, 寡胜肽膜透酶可調控 A 群鏈球菌許多致病與調節因子的表現, 且對 BALB/c 小鼠具較高致死率。 其次, Opp 可引發宿主之訊息傳遞以增加 A 群鏈球菌附著、 侵入與穿透細胞層能力。
  Group A streptococcus (GAS) is an important human pathogen that causes pharyngitis, toxic shock syndrome and many other human respiratory tract and tissue infections. The oligopeptide permease (Opp) of GAS is a membrane-associated protein and belongs to the ABC-transporter family. It is coded from a polycistronic operon containing oppA, oppB, oppC, oppD and oppF. The biological function of these genes is not clear in GAS. To understand the role of Opp on streptococcal infection, an isogenic oppA mutant was constructed by using an integrative plasmid to disrupt the opp operon and confirmed by Southern blot hybridization and RT-PCR. The oppA isogenic mutant not only decreased transcription of speB, speX, and rofA, but also increased transcription of speF, sagA (streptolysin S associated gene A), slo (streptolysin O), pel (pleotrophic effect locus), and dppA (dipeptide permease). No effects were found on transcription of emm, speJ, speG, rgg, and csrR. The oppA mutant caused more dissemination and less mortality and tissue damage than the wild-type strain when inoculated into BALB/c mice. The efficiency of adhesion to and internalization by host cells decreased about 50% and 46%, respectively, in opp mutant strain compared with those of the wild-type strain. The pull down and western blot assays showed that Opp may trigger the Rac, Rho activation, FAK and JNK phosphorylation of A549 cells and caused cytoskeleton rearrangement after GAS infection. Immunoflouresence staining and transmission electromicroscopy demonstrated that Opp mediated the tight junction open in A549 and MDCK cells. This would lead to open the cell tight junction that enhances GAS entry to deeper tissue. Based on these data, we conclude that the opp operon plays two important roles in the pathogenesis of group A streptococcal infection. First, Opp can regulate several important streptococcal virulence factors and regulators which would lead to severe mortality in BALB/c mice. Second, Opp can trigger host signal transduction that enhances streptococcal adhesion, internalization and invasion.
目 錄 Page
中文摘要 i
英文摘要 ii
誌謝
表目錄 vi
圖目錄 v
前言 1
材料與方法
I. 本研究各實驗之藥品、 溶液配方及儀器 32
II. 菌種、 質體、 細胞株及實驗動物來源 32
III. 菌種與細胞株培養與保存 32
IV. 生長曲線測試 32
V. 大腸桿菌質體 DNA 之抽取 33
VI. A 群鏈球菌質體 DNA 之抽取 33
VII. A 群鏈球菌染色體 DNA 之抽取 34
VIII. 質體 DNA 的選殖 34
IX. 大腸桿菌勝任細胞(Competent cell)的製備 34
X. 大腸桿菌細胞轉型作用(Transformation) 35
XI. A 群鏈球菌電穿孔轉型作用(Electroporation) 35
XII. 菌落及南方墨漬雜交法( Colony and Southern
blotting hybridization)
36
XIII. A 群鏈球菌 RNA 之抽取 39
XIV. 北方墨漬法 (Northern blot assay) 40
XV. 聚合酶連鎖反應(Polymerase chain reaction;PCR) 41
XVI. Reverse transcriptase PCR (RT-PCR) 分析 41
XVII. 蛋白質酶活性測試 42
XVIII. SLO 溶血素 (hemolysin) 活性測定 42
XIX. SLS 溶血素 (hemolysin) 活性測定 43
XX. A 群鏈球菌莢膜中玻尿酸(Hyaluronic acid)含量測

43
XXI. A549 表皮細胞總量蛋白質萃取 (whole cell protein
extraction)
44
XXII. 蛋白質濃度定量 44
XXIII. SDS-PAGE 蛋白質膠體電泳 44
XXIV. 西方點墨法 (Western blotting) 45
XXV. SpeB 多元抗體 (polyclonal antibody) 與 A 群鏈球
菌抗血清 (antiserum) 之製備
45
XXVI. A 群鏈球菌動物感染模式 (Air pouch model of
infection) 與 LD50 測試
46
XXVII. A549 上皮細胞之 DNA transient transfection 47
XXVIII. A 群鏈球菌附著細胞能力 (adhesion) 測試 47
XXIX. A 群鏈球菌侵入細胞能力 (Internalization) 測試 48
XXX. A 群鏈球菌穿透細胞能力 (Invasion) 測試 48
XXXI. 免疫螢光染色 (Immunofluorescence staining) 49
XXXII. 利用 Pull-down assay 收集活化態 Rho family
GTPase (Rac, Rho 和 CDC42)
50
XXXIII. 電子顯微鏡之樣品製備與觀察 51
XXXIV. Bactericidal assay 52
XXXV. A 群鏈球菌 in vivo dissemination 52
XXXVI. Cell viability assay 53
XXXVIII. 影像處理與統計方法
53
結果
A. 利用菌落雜交法篩選寡胜肽膜透酶 (oligopeptide
permease, Opp)
54
B. A 群鏈球菌 opp 突變株 (SW552) 、互補株
(SW563)、 回復株 (SW553) 之篩選
B-1. opp 突變株之構築 55
B-2. opp 回復突變及互補株之構築 56
B-3. opp 野生株及各突變株 protease 活性之測試 57
B-4. opp 野生株及突變株 oppA RT-PCR 之測試
57
C. A 群鏈球菌 opp 基因對致病因子的調控
C-1. opp 對 streptococcal exotoxins 的影響 58
C-2. opp 對 streptococcal virulence factors 的影響 58
C-3. opp 對 streptococcal regulators 的影響 59
D. Opp 對 A 群鏈球菌附著、 侵入及穿透能力的影響
D-1. opp 對其它 streptococcal adhesins 表現的影響 60
D-2. opp 對宿主細胞細胞骨架重組的影響 61
D-3. opp 對宿主細胞訊息因子活化的影響
D-3-1. opp 與 Rho subfamily of small GTPase 的關係 63
D-3-2. Opp 與 Rho GTPase 在 A 群鏈球菌附著過程中之
關連性
63
D-3-3. Opp 與 FAK 在 A 群鏈球菌附著過程中之關連性 66
D-3-4. Opp 與 MAPK 在 A 群鏈球菌附著過程中之關連性 67
D-4. A 群鏈球菌藉由 Opp 引發細胞緊密接合 (tight
junction) 破壞而穿透細胞層
68
E. Opp 參與 A 群鏈球菌在 BALB/c 小鼠體內之
dissemination
70
F. Opp 對 A 群鏈球菌感染 BALB/c 小鼠之影響
70
討論
A. 利用生物資訊學對 A 群鏈球菌寡胜肽膜透酶之功
能探討
73
B. 寡胜肽膜透酶與 A 群鏈球菌致病因子相關性探討
B-1. 寡胜肽膜透酶與 A 群鏈球菌調控因子之關係 75
B-2. 寡胜肽膜透酶與 A 群鏈球菌溶血素之關係 76
B-3. 寡胜肽膜透酶與 A 群鏈球菌外毒素之關係
77
C. A 群鏈球菌寡胜肽膜透酶與細胞附著、 侵入及穿
透功能之探討
C-1. 寡胜肽膜透酶與A 群鏈球菌對細胞附著能力之相
關性
78
C-2. 寡胜肽膜透酶與A 群鏈球菌對細胞侵入能力之相
關性
C-2-1. 寡胜肽膜透酶與宿主細胞之 small GTPase 活化的
關係
80
C-2-2. 寡胜肽膜透酶與宿主細胞之focal adhesion kinase
活化的關係
82
C-2-3. 寡胜肽膜透酶與宿主細胞之 MAPK 活化的關係 82
D. 寡胜肽膜透酶與A 群鏈球菌穿透細胞層能力與在
體內散佈之相關性
84
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86

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