酵母菌臨床分離株高溫生長的調控機制

Abstract

酵母菌Saccharomyces cerevisiae在過去一直被認定為安全且不具感染性的單細胞真菌，也廣泛用於釀酒及麵包發酵等食品工業上，然而自從1980年代起，在免疫力低落及癌症等病人身上陸續發現有S. cerevisiae菌株感染的案例，1990年起也有研究團隊針對菌株在小鼠中的感染能力區分菌株的致病性，1994年研究也發現菌株的致病性與其能否高溫生長有高度的相關，然而對於這些臨床具感染性菌株為何具有42℃高溫生長特性至今仍未清楚證實，因此本次研究利用兩個台大臨床分離株的子菌株(YYC870/YY871)與實驗室菌株(YYC861)進行一系列高溫生長相關性狀及基因表現的探討，期望從中找出臨床致病菌株高溫生長調控標的以作為後續臨床檢測及治療之應用。我們從固態培養基5天42℃培養的結果證實臨床菌株可在42℃下生長，而實驗室菌株則否，過去研究也指出高溫引發的氧化壓力可能是影響細胞生長及生存的重要因素，因此，我們將高溫培養的菌株以DHE (Dihydroethidium)分析菌株累積活性氧的情形 我們發現經持續高溫培養，臨床菌株相較於實驗室菌株累積較低的活性氧，此外，以抗氧化劑(NAC, N-Acetyl-L-cysteine)作用於實驗室菌株也看到活性氧累積降低及恢復高溫生長的性狀，以上實驗結果證實高溫下持續累積的活性氧將使菌株高溫生長受到抑制進一步危及到菌株的生存，從過去研究得知，活性氧累積一方面會啟動細胞抗氧化機制以降低活性氧持續累積，另一方面，過度累積的活性氧也會破壞細胞的脂質、蛋白質、DNA結構及功能，促使細胞啟動相關的修復機制以維持正常生理功能。因此，我們藉由TUNEL assay的方式分析菌株在高溫生下DNA斷裂的情形，實驗結果發現在高溫培養5天實驗室菌株DNA斷裂情形明顯高於臨床菌株，另外，也根據實驗室過去對於臨床菌株及實驗室菌株，37℃高溫生長相較於30℃生長下，以Tiling array比較結果中發現，DDR2 (DNA Damage Responsive)這個基因在臨床菌株高溫下誘發的表現量高於實驗室菌株，DDR2在過去研究指出在具氧化及高溫壓力下會高度表現，由以上結果我們推論DDR2在臨床菌株高溫下的高表現可能具有較高的修復能力，使臨床菌株得以維持細胞正常生理功能。總結以上結果，本研究證實臨床菌株與實驗室菌株在高溫下活性氧累積的差異及誘發細胞基因表現的不同可作為分析臨床菌株高溫生長差異、致病性與否的重要依據。

The yeast Saccharomyces cerevisiae has been identified as safe and non-pathogenicity fungus in the past, also widely used in the brewing , fermentation of bread and other food industries. However, since 1980s, S. cerevisiae have been discovered in cases with low immunity or in cancer patients. Since 1990, some research teams focused on the infectivity of S. cerevisiae in mice to distinguish pathogenic and non- pathogenic strains. In1994, some studies also found high correlation of strain pathogenicity and the ability of high temperature growth. However, it is not yet clear that why these infective clinical strains has high temperature growth ability at 42 ℃. In this study, we investigated a series of high-temperature growth-related traits and gene expression by characterizing the differences between two segregants of NTU clinical isolates (YYC870/YY871).We expect to find out the target of high temperature growth regulation in clinical pathogenic strains as the application of subsequent clinical diagnosis and treatment. First, we confirmed the different ability to grow at 42 ℃ between clinical isolates and laboratory strains. The results from culture on the solid medium for 5 days at 42 ℃ indicated that clinical strains grow at 42 ℃ but laboratory strains don’t. Previous study pointed out that high-temperature-induced oxidative stress may be an important factor affecting cell growth and survival. Therefore, we analyzed reactive oxygen species accumulation in cells growing at high temperature. In addition, treating antioxidants (NAC, n-Acetyl-L-cysteine) reduced reactive oxygen species accumulation and restored high temperature growth in the laboratory strain. According to the results , we confirmed that ROS accumulation at high temperature inhibited strains growth and further threaten their survival. Previous study also showed that cells activate antioxidant mechanisms to reduce accumulation of reactive oxygen species when growing at high temperature; on the other hand, excessive accumulation of reactive oxygen species also damage lipids, proteins, or DNA structure and promote cell repair mechanisms to maintain normal physiological function. Therefore, we analyzed DNA fragmentation in cells at high temperature by TUNEL assay. The experimental results showed that DNA fragmentation levels were significantly higher in laboratory strains than clinical isolates for 5 days cultured at high temperature. In addition, based on the previous Tiling array data, we found induced higher expression of DDR2 (DNA Damage Responsive) in clinical isolates than in laboratory strains. Previous studies showed DDR2 expression was significantly increased in oxidative stress or at high temperature .We assumed DDR2 in clinical strains may be the key player for repair. Therefore, clinical isolates maintain the normal physiological function under high temperatures .Summing up the above findings, our study confirmed the differences between clinical isolates and laboratory strains in reactive oxygen species accumulation and gene expression. This can be a basis on analysis of clinical strains of high temperature growth and their pathogenicity.