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研究生(外文):Pei-Chi Chiang
論文名稱(外文):A Comparison of Cell Wall Proteomics between Clinical and Laboratory Saccharomyces cerevisiae Strains by Mass Spectrometric Analysis
中文關鍵詞:Saccharomyces cerevisiae致病性細胞壁蛋白質質譜分析SILAC
外文關鍵詞:Saccharomyces cerevisiaepathogenicitycell wall proteinmass spectrometric analysisSILAC
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酵母菌Saccharomyces cerevisiae普遍被使用於食品工業、生物科學研究,然而近年來臨床感染報告的增加,使其成為一個可能的新興真菌病原,本研究藉由比較臨床分離菌株與實驗室菌株之間的差異,探討臨床菌株可能的致病機轉。首先希望建立一個體外檢測S. cerevisiae致病性的方法,能夠在進行活體實驗之前對菌株致病性做一個初步的篩選,過去研究指出S. cerevisiae菌株致病性和其刺激巨噬細胞產生促發炎細胞激素的能力呈正相關,我們利用菌株感染BALB/c及C57BL/6小鼠腹腔分離的巨噬細胞,發現其TNF-α的分泌量和菌株致病能力之間無法歸納出關聯性,顯示此體外檢測系統的可行性需要再評估。真菌細胞為細胞壁所包覆,細胞表面結構的改變將影響菌株和免疫細胞的交互作用,以MATH (Microbial Adhesion To Hydrocarbon)方法測定菌株細胞表面疏水性,結果顯示相較於實驗室菌株,臨床菌株表現出較高的疏水性,不同菌株之間細胞表面的性質確實有所不同。酵母菌細胞壁的最外層是由許多緊密堆疊的甘露醣蛋白所組成,利用質譜分析配合SILAC (Stable Isotope Labeling by Amino acids in Cell culture)定量方法比較臨床菌株和實驗室菌株細胞壁蛋白質體的組成,結果顯示S. cerevisiae臨床菌株整體細胞壁蛋白質的表現量較實驗室菌株為高,尤其以Scw10p、Pst1p、Hsp150p三個蛋白質最為顯著,這些蛋白質和臨床菌株致病性的關係期待更多的研究來探討。我們推測臨床菌株表現較多的細胞壁蛋白質,而能在細胞外層緊密地堆疊成為一面屏障,將細胞壁內層易引發免疫反應的β-glucan隱藏起來,免疫系統因而無法偵測其存在,使之能夠在宿主體內停留較長時間而不被清除,但此推測仍有待進一步證實;總結來說,本研究發現S. cerevisiae臨床菌株和實驗室菌株細胞壁蛋白質體的差異,這可能有助於釐清臨床菌株表現出較高致病能力的原因。
The yeast Saccharomyces cerevisiae is widely used in food industry and biotechnology. However, it has been regarded as an emerging fungal pathogen because of an increasing number of infections being reported in these two decades. In this study, we investigated the possible pathogenic mechanism of S. cerevisiae by characterizing the differences between the clinical and laboratory strains. First, we tried to set up an in vitro system of virulence determination, by which we can get some preliminary concepts of pathogenic potential of the yeast strains before in vivo murine model experiment. Previous study revealed that the virulence of S. cerevisiae strains correlated with the potential of proinflammtory cytokine induction in macrophages. We used the isolated peritoneal macrophages from BALA/c and C57BL/6 mice as the infected cells to do this test. However, we did not observe the correlation between TNF-α production levels and the pathogenicity of the clinical isolates we collected. The feasibility of the in vitro system should be further investigated. Fungal cells are surrounded by a cell wall, alterations of which are known to influence the host immune response after infection. MATH (Microbial Adhesion To Hydrocarbon) was used to characterize the cell surface property of different yeast strains, and the results showed that the clinical strains are more hydrophobic than the laboratory strains, indicating certain difference existed on the cell surface between these strains. The outermost part of yeast cell wall is composed of a dense layer of mannoproteins. To examine the difference in cell wall protein composition between the clinical and laboratory strains, we performed a mass spectrometry-based quantitative proteomics with the labeling strategy using SILAC (Stable Isotope Labeling by Amino acids in Cell culture). The expression levels of most cell wall proteins increased in the clinical strains we observed, especially for proteins Scw10p, Pst1p, and Hsp150p. Further study should be done to understand whether these proteins play important roles in the pathogenicity of the clinical strains. We propose that the clinical strains upregulate cell wall protein expression to form a dense coat, under which the potent proinflammatory β-glucan of inner cell wall cannot be recognize by host immune system. Thus, the yeast cells are capable of prolonged persistence in the host. However, this hypothesis needs further confirmation. Altogether, we reported here the characterization of the difference in cell wall proteomics that might be important to the pathogenicity of the clinical strains.
中文摘要 I
英文摘要 II

緒論 1
材料與方法 7
一、實驗菌株與培養方式 7
二、S. cerevisiae菌株致病性的體外初步篩選 7
1. 小鼠腹腔巨噬細胞的分離 7
2. 小鼠腹腔分離巨噬細胞之純度檢測 9
3. S. cerevisiae菌株對數期細胞的取得 9
4. S. cerevisiae菌株感染前處理 10
5. 菌株感染與細胞激素的誘導 10
6. TNF-α分泌量的測定 11
三、S. cerevisiae細胞表面疏水性的測定 12
1. S. cerevisiae菌株對數期細胞的取得 12
2. MATH (Microbial Adhesion To Hydrocarbon) 12
3. 以黏附到o-xylene的細胞比例表示S. cerevisiae細胞表面的疏水性 13
四、S. cerevisiae臨床分離株及實驗室菌株細胞壁蛋白質體的比較 13
1. 建構賴氨酸合成路徑有缺失的雙倍體S. cerevisiae實驗室菌株 13
2. S. cerevisiae菌株對數期細胞的取得及穩定同位素標定胺基酸的嵌入 14
3. 臨床分離株/YYC370細胞壁的分離 15
4. 質譜樣品的前處理 16
5. 奈米毛細管液相層析串聯質譜儀分析(nano-LC-MS/MS) 17
6. 質譜結果的定量分析 18
實驗結果 19
一、S. cerevisiae菌株致病性體外篩選系統的建立 19
二、S. cerevisiae菌株細胞表面疏水性的測定 21
三、以質譜分析S. cerevisiae的細胞壁蛋白質體 22
四、細胞壁蛋白質SILAC相對定量系統的建立 23
五、S. cerevisiae臨床菌株和實驗室菌株細胞壁蛋白質的相對定量 25
討論 26
圖表 32
參考資料 49
附錄 54
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