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研究生:何俊達
研究生(外文):Chun-Ta Ho
論文名稱:轉糖鏈球菌對於人類血漿刺激的基因表現分析
論文名稱(外文):Genome-wide expression analysis of human plasma-regulated genes in Streptococcus mutans
指導教授:賈景山
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:微生物學研究所
學門:生命科學學門
學類:微生物學類
論文種類:學術論文
論文出版年:2008
畢業學年度:97
語文別:中文
論文頁數:70
中文關鍵詞:轉糖鏈球菌感染性心內膜炎血漿微陣列分析雙分子調控系統過氧化氫檸檬酸發酵作用
外文關鍵詞:Streptococcus mutansinfective endocarditisplasmamicroarraytwo-component systemhydrogen peroxidecitric acidfermentation
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轉糖鏈球菌是屬於人體口腔內正常的常態菌落,在口腔內的環境時可以代謝各種不同的醣類,代謝後的酸性產物會引起龋齒;而當口腔有傷口時,轉糖鏈球菌可以轉型成為伺機型病菌,在血液中沿著血管循環全身,並在受損的心臟瓣膜形成感染性心內膜炎。在從口腔經由血液抵達心臟瓣膜後最後形成疾病的過程中,最重要的毒性因子應該屬於具有黏附性質的黏附蛋白質和可以維持細菌持續生長的基礎代謝基因所轉錄出的蛋白質。本研究採取兩種不同的實驗方法,試圖尋找出共同調控的毒性因子,包括利用了微陣列分析受血漿刺激的轉糖鏈球菌和篩選對於存活於體內重要的雙分子調控系統之調控子。微型陣列分析結果顯示總共有206個基因的表現量提高或下降,根據基因的功能分類這些基因,受到血漿刺激影響最多的功能族群是未知功能的基因,約有30%;其次是能量基礎代謝的基因,約有20%;第三個則是關於已知毒性因子基因的族群,約有12%。在未知功能的蛋白質當中,有一些被認為具有分泌到外界或表現在細菌細胞膜表面上的能力;而在能量基礎代謝的基因中,則觀察到有許多有關醣類代謝以及運輸幫浦的基因表現量上升,同時也觀察到發酵作用相關的基因表現量上升;在毒性因子方面則觀察到有與黏附作用有關的蛋白質,如:glucosyltransferase B和D的表現量改變。本研究也同時觀察到預先以血漿刺激S. mutans30分鐘或60分鐘後,可以提高轉糖鏈球菌在嗜中性多核球或在過氧化氫 ( H2O2 ) 刺激後的存活率,暗示了血漿除了可以調控基因來適應環境外,也可以改變外在表現型來存活於血液當中。在利用大鼠篩選雙分子調控系統的調控子突變株的實驗中,本研究觀察到在感染大鼠30分鐘後已有三個突變株顯現出生存能力降低之趨勢,分別為RR6、RR8、RR10,其中RR8對於H2O2的敏感程度高於野生株,因此本研究更進一步去分析此一突變株附近之基因組成, 觀察到RR8突變位置附近的基因與微型陣列分析的基因重複,而這些基因主要以分解檸檬酸為主。根據以上研究結果,本研究推測轉糖鏈球菌在血漿生長時可能利用另類碳源,進行發酵作用產生ATP,並且可以偵測血漿中的物質來加強在嗜中性多核球存活的能力或抵抗來自過氧化氫的攻擊。
Streptococcus mutans which is a commensal in human oral cavity can metabolize various kinds of sugars and the end product of these metabolisms is the leading cause of dental caries. Being an opportunistic pathogen, S. mutans sneaks into blood steam through injury in oral cavity, circulates in the blood and adheres to damaged heart valve causing infective endocarditis. The pathogenesis of infective endocarditis of S. mutans relies on two important virulence factors: adhesins and carbohydrate-metabolic proteins. Genes transcribed adhesins help bacteria to bind to damaged valve and carbohydrate-metabolic genes sustain the growth of bacteria in human blood. In this study, two experimental strategies were used to elucidate the co-regulated virulence factors in S. mutans; identifying plasma-regulated genes by microarray and screening two component system ( TCS ) essential for in vivo survival. The microarray analysis indicated that among a total of 206 genes up- or down- regulated after 60 minutes exposure to human plasma, 30% of them were hypothetical proteins, 20% of genes involved in energy metabolism and 12% of them were putative virulence factors. Some plasma-regulated hypothetical proteins are predicted to be secreted or membrane anchored; most up-regulated genes of energy metabolism belong to sugar metabolism, transportation or fermentation; and some previously identified virulence factors, such as genes encoding putative adhesion protein, glucosyltransferase B and D were also identified. Prior-exposure of S. mutans to plasma for 30 or 60 minutes could enhance the ability of bacteria to survive killing in neutrophil or H2O2 treatment, suggesting that human plasma might alter phenotypical characteristics in S. mutans in addition to changes in gene transcription in order to survive in the blood circulation. The in vivo clearance screening identified three TCS mutants: RR6, RR8 and RR10 which are attenuated in survival. Among these mutants, only RR8 exhibited increased sensitivity to hydrogen peroxide. Some of the plasma-regulated genes, mainly citrate degradation related, are located near-by the TCS identified by the RR8 mutant. It is possible that S. mutans might utilize alternative carbon resources to generate ATP through fermentation when growing in plasma and bacteria could sense plasma components to enhance survival in neutrophil or ability to counteract H2O2.
口試委員會審定書 ii
致謝 iii
目 錄 iv
圖目錄 vi
表目錄 vii
中文摘要: viii
英文摘要: x
第一章 緒論: 1
第一節、口腔內的菌叢和轉糖鏈球菌之分類 1
第二節、轉糖鏈球菌之基因體特色 2
2.1 基因體概觀 2
2.2 基礎代謝基因概觀 2
2.3 毒性因子基因概觀 3
第三節、轉糖鏈球菌引起之疾病 5
3.1 齟齒 5
3.2 感染性心內膜炎 5
第四節、致病菌與人體血液互動之關係 6
第五節、毒性因子與參與基礎代謝之基因 8
第六節、實驗目的及設計: 10
第二章 實驗材料與方法: 11
一、菌株 11
二、藥品與材料 11
三、轉糖鏈球菌之培養 11
四、轉糖鏈球菌對過氧化氫的敏感度試驗 12
五、純化嗜中性多核球 12
六、嗜中性多核球吞噬轉糖鏈球菌試驗 12
七、活體篩選轉糖鏈球菌調控子突變株 13
八、聚合酶鏈鎖反應 13
九、萃取轉糖鏈球菌之核酸 13
十、微型陣列雜交反應 14
十一、微型陣列之資料分析 16
第三章 實驗結果: 17
第一節、篩選毒力下降之調控子突變株 17
第二節、血漿刺激可協助轉糖鏈球菌在嗜中性多核球中生存 17
第三節、血漿刺激可降低轉糖練球對於過氧化氫之敏感程度 18
第四節、觀察血漿對於轉糖鏈球菌在整體轉錄層面上的影響 19
第五節、受到血漿刺激而提高其表現量的基因 20
第六節、受到血漿刺激而降低其表現量的基因 25
第七節、毒力減弱之突變株與血漿調控基因之關係 31
第八節、毒力減弱突變株對於過氧化氫敏感度試驗 32
第四章 討論: 33
參考文獻: 59
附圖 69
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