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  • 學位論文

探討台灣臨床分離的菌株Saccharomyces cerevisiae主要的致病因子之一:高溫生長能力

Analysis of the ability of high temperature growth, one of the major virulence traits, in clinical isolate of Saccharomyces cerevisiae in Taiwan

指導教授 : 張雅雯

摘要


Saccharomyces cerevisiae一般來說不具有致病性,但近年來陸續感染病例的報告,所以被認為也是一種伺機性感染的病原體。先前的研究指出釀酒酵母菌的臨床菌株多出了能在高溫(42°C)下生長以及假菌絲產生的能力,推測這兩者為可能的致病因子。在台大醫院分離之三個臨床菌株,我們發現當中有兩個具有高溫生長能力以及假菌絲生成能力。本研究目標主要為探討S. cerevisiae的高溫生長能力這個致病因子。先前研究指出高溫生長屬多基因調控,可能參與的基因有:MKT1、RHO2、END3,另外也發現在這些基因上若帶有某些SNP會影響到高溫生長的表現型。我們發現具有高溫生長能力的臨床菌株的定序結果,發現在MKT1 (A1057G)和RHO2 (T271G)處的SNP會造成胺基酸改變,而在這些位置上的等位基因屬異型合子(heterozygous),但沒有高溫生長能力的菌種則無。於是進行四分孢子分離,得到了同型合子的子代。而分離出來的子代中發現只有一株具有和母代一樣強的高溫生長能力,我們將此子代的高溫生長基因(MKT1)送入實驗室菌株中,觀察高溫生長的表現型是否會因此而有所改變,但經此改造的實驗室菌株,其高溫生長能力並未有發現有顯著的變異,這表示只單純改變一個高溫生長基因,並未能夠影響到其表現型。當酵母菌受到高溫壓力之時,細胞內活性氧化自由基會上升,若此時細胞抗氧化壓力的能力不足,細胞就無法存活下去。我們去比較抗高溫的菌株和不耐熱的菌株比較在受到高溫壓力之時其細胞內活性氧化自由基的累積情況,但結果並未發現抗高溫的菌株其活性氧化自由基的累積會比較低。目前我們尚未找到抗高溫生長的關鍵因子,之後仍需進一步的研究。

並列摘要


Saccharomyces. cerevisiae has recognized as “generally regarded as safe” organism, but recent evidence indicated that the clinical isolates were virulent and had been implicated in the induction of a disease. The pathogenic isolates exhibit the ability to grow at 42°C and are capable of pseudohyphal growth. Our lab got three clinical isolates(YYC1、YYC2、YYC3) from NTUH . We found there were two of them having the ability of growth at 42°C and pseudohyphal formation. My target is studying the ability of growth at 42°C. The previous report mapped a S. cerevisiae high temperature growth QTL and identified MKT1, END3, and RHO2 as QTGs. There were some important SNPs (ex.MKT1 A1358G、END3 C733T )contributing to HTG phenotype. In our case, we found that the SNPs in YYC1, MKT1 (A1057G) and RHO2 (T271G), are nonsynonymous changes and heterozygous in these two gene locus. Next, we use tetrad dissection, making the homozygous segregants, try to observe the contribution of these SNPs to HTG. After tetrad dissection analysis, we found that there were few segregants(4/60) having the HTG phenotype. Then, we cloned the MKT1, from one of the segregants which had thermo-tolerance, into a mkt1 deletion strain. However, the clone did not change its phenotype. This indicated that the single genotype alternation did not affect the HTG phenotype. When the yeast cells encounter heat stress, the cellular ROS (reactive oxygen species) will be induced. Excess ROS causes damages by attacking DNA and proteins. Our hypothesis was that the thermo-tolerant strains might have more strong ability to clear up cellular ROS. But the experiment result indicated that the thermo-tolerant strains did not have lower ROS level than thermo-sensitive strains. We still do not find the key factor contribution to the ability of HTG.. It needs advanced researches to figure out the mechanism of high temperature growth.

參考文獻


Aucott, J. N., J. Fayen, et al. (1990). "Invasive infection with Saccharomyces cerevisiae: report of three cases and review." Rev Infect Dis 12(3): 406-11.
Borkovich, K. A., F. W. Farrelly, et al. (1989). "hsp82 is an essential protein that is required in higher concentrations for growth of cells at higher temperatures." Mol Cell Biol 9(9): 3919-30.
Burnie, J. P., T. L. Carter, et al. (2006). "Fungal heat-shock proteins in human disease." FEMS Microbiol Rev 30(1): 53-88.
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Clemons, K. V., J. H. McCusker, et al. (1994). "Comparative pathogenesis of clinical and nonclinical isolates of Saccharomyces cerevisiae." J Infect Dis 169(4): 859-67.

被引用紀錄


詹佳璇(2013)。篩選黃芝酒精萃取物延長酵母菌壽命及抗老化機制探討/探討細胞壁蛋白質Hsp150p在台灣酵母菌臨床分離菌株的表現型〔碩士論文,國立臺灣大學〕。華藝線上圖書館。https://doi.org/10.6342%2fNTU.2013.02069

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