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標題: | 以蛋白質體學分析轉糖鏈球菌表面蛋白質受血漿成份誘發之表現 Proteomic analysis of Streptococcus mutans surface proteins induced by plasma components |
作者: | Ya-Hsiung Shieh 謝亞雄 |
指導教授: | 賈景山(Jean-San Chia) |
關鍵字: | 轉糖鏈球菌,心內膜炎, S. mutans,endocarditis, |
出版年 : | 2005 |
學位: | 碩士 |
摘要: | 草綠色鏈球菌 (viridans streptococci) 和葡萄球菌 (staphylococci) 是造成感染性心內膜炎最主要的致病菌。有能力在血漿中存活並且在受損的心臟瓣膜處聚集,為感染性心內膜炎 (infective endocarditis) 的主要致病因子。然而,細菌能夠適應不同環境壓力對於自身的存活以及致病能力是非常重要的。草綠色鏈球菌表面蛋白質與血漿成份之間的互動,與血小板凝集及受傷心瓣膜的附著皆扮演重要的角色。在本實驗中轉糖鏈球菌的表面蛋白質以二維電泳分開,經過胰蛋白質?分解後,以 Matrix-Assisted Laser Desorption Ionization with Time of Flight (MALDI-TOF) 質譜儀定出二維電泳膠上 63 個蛋白質點。在轉糖鏈球菌 UA159 基因體上得到 55 個不同的 open reading frames (ORFs) 。為了驗證不同的環境壓力對於轉糖鏈球菌表面蛋白質表現的影響。我利用二維電泳分析轉糖鏈球菌表面蛋白質在不同酸鹼值的環境下,及含有血漿成份的生長情況下的表現差異。多個表面蛋白質都受到環境酸鹼值及血漿成份的調控,其中包括了 N-acetylmuramidase 和 extracellular glyceraldhyde-3-phosphate dehydrogenase (eGAPDH) 。進一步發現轉糖鏈球菌與內皮細胞的結合能力在含有血清成份中會增加。為了找出能夠與細菌表面結合的血漿成份,我將轉糖鏈球菌加入血漿結合後直接以二維電泳和質譜儀分析結合情形。我成功得到了 30 個能與轉糖鏈球菌結合的血漿成份,而纖維蛋白質原也包含在內。在心內膜炎的動物實驗中發現,同時含有纖維蛋白質原及纖維結合素結合能力對金黃色葡萄球菌在心瓣膜上聚集以及入侵內皮細胞相當重要。我利用二維電泳結合遠西方墨點法分析轉糖鏈球菌的纖維結合素結合蛋白質及纖維蛋白質原結合蛋白質。我發現兩個轉糖鏈球菌的表面蛋白質, N-acetylmuramidase 和 eGAPDH ,同時具有纖維結合素與纖維蛋白質原的結合能力。綜合實驗結果我證實血漿成份的確會影響轉糖鏈球菌表面蛋白質的表現,而且附著在細菌表面的血漿蛋白質也有助於細菌在內皮細胞上的吸附作用。 Viridans group streptococci and staphylococci are the pathogens most frequently responsible for infective endocarditis. The ability of bacteria to survive in plasma and colonize damaged heart valves are essential for inducing infective endocarditis. We hypothesize that interaction of viridans streptococci surface protein with components of the plasma may induce adaption of bacteria and contribute to the virulence in infective endocarditis. Cell wall-associated proteins from Streptococcus mutans are extracted and separated by two-dimensional gel electrophoresis (2-DE), following by trypsin digestion and Matrix-Assisted Laser Desorption Ionization with Time of Flight mass spectrometry (MALDI-TOF MS) analysis. A total of 63 major spots is analyzed and among them 55 exhibite distinct identity based on the protein data base deduced from S. mutans UA159. Changes in the S. mutans surface protein profiles in response to external pH shift or addition of plasma components are subsequently analysed from cells culturing under chemostat conditions. I identify several new protein spots that appear after a pH shift of the bacteria from 5.5 to 7.3 or exposing the bacteria to plasma components. The proteins that appear after the treatments include N-acetylmuramidase and extracellular glyceraldhyde-3-phosphate dehydrogenase (eGAPDH). We have also demonstrated that the binding of S. mutans to endothelial cells is significantly enhanced by pre-incubating of the bacteria with serum, suggesting that serum or plasma proteins are cracial to the binding of bacteria to the endothelial cells. To identify the identities of the plasma proteins bound to bacteria in vitro, that interact with bacteria plasma proteins are co-eluted with S. mutans surface proteins and analyzed by 2-DE and MALDI-TOF MS, which identify about 30 plasma proteins, including fibrinogen. 2-DE and far-western find that N-acetylmuramidase and eGAPDH of S. mutans interact with fibrinogen and fibronectin. According to these results, I conclude that external environmental stress induces changes the outer surface protein profiles of S. mutans, and such changes in collaboration with plasma proteins may enhance the binding of bacteria to endothelial cells. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/35752 |
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