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

Abstract

摘 要 – 第一部分 探討酵母菌壽命分析方法為複製存活壽命(replicative lifespan；RLS)，定義為計算母細胞所分離出的子代數作為其壽命數，時序壽命(chronological lifespan；CLS)，定義為測定細胞培養在液態培養液中存活時間作為其壽命數。目前細胞老化理論指出細胞內活性氧(reactive oxygen species；ROS)增加是造成細胞壽命減短的因素。科學家亦藉由探討天然小分子例如:橙皮柑(hesperidin)延長酵母菌壽命機制，釐清細胞內老化路徑。靈芝屬(Genus Ganoderma)屬於真菌界生物，先前研究指出靈芝粗萃取物具有抗氧化功能，但是目前尚無研究探討靈芝的抗氧化功能是否具有延長壽命的證據。本實驗目的即以RLS篩選靈芝屬粗黃芝(Ganoderma colossum)的萃取物EEGC是否具有使酵母菌壽命延長效果，再分析EEGC在酵母菌內的抗氧化效果，最後探討其抗氧化能力是否參與抗老化機制。實驗首先測試菌株(BY4742)生長曲線實驗確定EEGC是否具酵母菌生長抑制，結果顯示2 mg/ml EEGC處理下酵母菌生長情形較低，而選用0.5及1 mg/ml EEGC處理酵母菌。 N-乙醯基半胱氨酸(N-acetylcysteine；NAC)具有解除細胞內氧化壓力的功能，作為RLS實驗陽性控制組，RLS實驗觀察發現0.5 mg/ml EEGC具有顯著性延長酵母菌壽命。另外以CLS分析0.5及1 mg/ml EEGC發現無顯著性使酵母菌壽命延長。接下來分析EEGC處理下酵母菌內的ROS累積情形，結果發現0.2, 0.5或1 mg/ml EEGC顯著性使H2O2刺激下累積的ROS量下降，且同時具有抑制H2O2 刺激產生ROS累積的效果。接著操作即時定量聚合酶連鎖反應(real-time PCR) 發現EEGC具有降低SOD1、SOD2及SIR2 mRNA表現量，而測試SIRT1活性分析得到0.5及1 mg/ml EEGC具有顯著性增加SIRT1酵素活性，推測EEGC可能具有增加酵母菌Sir2p活性並藉由此抗老化路徑達到延長壽命結果，故未來可針對EEGC的抗老化機制做深入探討，以釐清細胞內的老化路徑。 摘 要 – 第二部分 酵母菌廣泛分佈於自然界中並使用在工業及食品工業上，然而近期臨床報告指出在免疫能力低下患者身上分離出具有致病性的酵母菌，使得人們重新思考致病性酵母菌潛在危險。實驗室先前研究台灣酵母菌臨床分離菌株與實驗室菌株的細胞壁蛋白質體組成差異發現，臨床菌株的細胞壁蛋白質Scw10p、Hsp150p、Pst1p蛋白量相較於實驗室菌株有顯著增加。Hsp150p屬於Pir家族蛋白，為釐清Hsp150p在酵母菌臨床分離菌株的細胞壁含量增加是否與其致病性有關，而深入探討Hsp150p與致病性分析。本研究建構三株臨床hsp150缺陷菌株，先以細胞壁表現型分析得到臨床hsp150缺陷菌株相對於親代菌株在Calcofluor white及Congo red環境下生長情形慢但並未影響菌株生長能力，顯示Hsp150p確實影響細胞壁完整性使細胞需要更長時間來適應有壓力的環境。接著測試細胞表面疏水性及塑膠表面吸附能力發現，臨床hsp150缺陷菌株具有降低細胞表面疏水性，但不顯著性影響塑膠表面吸附能力，表示Hsp150p可能參與在細胞表現疏水性調控。最後以酵母菌刺激巨噬細胞Raw264.7細胞株分泌TNF-α量推測臨床hsp150缺陷菌株的致病性，結果得到臨床hsp150缺陷菌株相較於原臨床菌株更能刺激TNF-α分泌，推測可能因Hsp150p缺陷造成細胞壁不完整導致未知的致病因子裸露。本研究中探討Hsp150p對臨床菌株造成表現型的變化，以期在未來能有助於細胞壁在臨床菌株致病性的探討。

Abstract – Part I There are two methods to access the lifespan of yeast. One is called replicative lifespan (RLS) defined by the number of daughter cells produced by single mother cell. The other method called chronological lifespan (CLS) defined by how long yeasts survived in liquid culture. Nowadays, it is proposed that the cellular accumulation of reactive oxygen species (ROS) might reduce the lifespan of organism. According to previous studies, under the treatment of small natural molecule like hesperidin, the lifespan of yeast was extended by reducing oxidative pressure. Genus Ganoderma belong to kingdom Fungi, and show obvious effect of antioxidant activities. However, there is still no report clearly stated that Genus Ganoderma could extend the lifespan of an organism by the antioxidant effect. In this study, we analyzed EEGC extracted from Ganoderma colossum to understand its anti-aging effect and mechanism of yeasts. At first, we established the treatment of 10 mM N-acetylcysteine (NAC) as the positive control of RLS. We found that 2 mg/ml EEGC could lower the growth rate of yeasts. In addition, it was hard to analyze effect of >1 mg/ml EEGC because it revealed turbidity. It was observed that 0.5 mg/ml EEGC could significantly extent RLS of yeasts but not CLS. Moreover, treatment of 0.2, 0.5, or 1 mg/ml EEGC all showed antioxidant effect to reduce the accumulation of ROS levels induced by H2O2. Our data also showed that EEGC could significantly decreased SOD1、SOD2、SIR2 mRNA levels in cells. The SIRT1 activity assay implied that 0.5 or 1 mg/ml EEGC may enhance Sir2p activity in yeast. Altogether, we demonstrated that EEGC may extend yeast replicative lifespan, and could act as an antioxidant in yeast. The anti-aging mechanism of EEGC and the correlation of Sir2p activity should be further investigated. Abstract – Part II Saccharomyces cerevisiae is widely distributed in nature, and has become increasingly important in biotechnology and food industry. However, numerous cases of clinical infection caused by S. cerevisiae have been reported in recent years, considering S. cerevisiae as an emerging opportunistic pathogen. Cell wall is the first surface of the cell to encounter stresses from host defenses and environmental stresses. In addition, cell wall is responsible for yeast viability and adhesion ability to host. Our previous analysis of the composition of cell wall protein between clinical isolates and laboratory strains found that cell wall proteins Scw10p, Hsp150p, and Pst1p expressed at higher levels in clinical isolates. To clarify the role of Hsp150p in clinical isolates, we constructed three hsp150 deletion strains in clinical isolate background, and analyze the impact in cell wall integrity, adhesion ability, cell surface hydrophobicity, and virulence. We found that hsp150 deletion strains grew slower than parental clinical isolates under high dosage of Calcofluor white or Congo red. Secondly, hsp150 deletion in clinical isolates did not affect the adhesion ability or cell surface hydrophobicity. Lastly, we found that hsp150 deletion strains stimulated mouse macrophage cell lines to secret higher levels of TNF-α than parental clinical isolates, implying that deletion of Hsp150p may interrupt cell wall integrity and expose unknown virulence factors to macrophages. In this study, we found that Hsp150p may be responsible for, at least in part, the integrity of cell wall, and may play a role in yeast virulence.