Rhb1-TOR訊息路徑對白色念珠菌致病因子調控之研究

The Rhb1-TOR Signaling Pathway in Regulation of Candida albicans Virulence Factors

陳昱庭(Yu-Ting Chen)

指導教授 ： 藍忠昱

國立清華大學/生命科學院/分子與細胞生物研究所/博士(2013年)

摘要

白色念珠菌(C. albicans)為常見的伺機性真菌病原菌。在免疫功能低下的患者，白色念珠菌的存在可能帶給患者致命的威脅。而我們的研究是為了了解期其致病因子的調控機制。近年來，我們發現小G蛋白Rhb1與雷帕霉素標的蛋白(TOR)共組而成之訊息傳遞路徑會調控某些致病因子的表達，包括分泌性天冬胺酸蛋白質水解酵素2號(Sap2)以及低氮源引發的形態生成(morphogenesis)。
本論文由兩個部分組成。在第一部分中，我們利用點突變、盈綠螢光蛋白標的及雷帕霉素感受性試驗證明了Rhb1在膜上的位置對於其功能是重要的。在人類與分裂酵母菌上已經證明了與GTP結合之Rhb1可藉由間接的方式調控Tor1。本研究同樣發現白色念珠菌的Rhb1也是透過非直接的方式影響Tor1，同時和Tsc2 (Rhb1之小G蛋白活化蛋白)有遺傳相互作用。更進一步地，我們發現在缺乏Rhb1時，存在有大量的Tsc2會去抑制Ras1-依賴性訊息並且進一步導致低氮源所引起的形態生成產生缺陷。進一步的結果說明了Ras1也參與在TOR訊息傳遞路徑且可能位於Rhb1之上游或與之無關。
我們接下來把重心轉移到Rhb1-TOR訊息路徑以及外在環境條件的改變是如何去調控分泌性天冬胺酸蛋白質水解酵素2號的表達上。在非活細胞內的研究發現，分泌性天冬胺酸蛋白質水解酵素2號是表達量最高的分泌性天冬胺酸蛋白質水解酵素，它也是個重要的致病因子。本研究發現當白色念珠菌生長在以蛋白質為主要可用之氮源的環境時，其細胞的生長是與Rhb1調控Sap2之表達有所關連。其中調控過程包與許多TOR訊息路徑類的分子有關，包括Tor1酵素本身與其下游之作用蛋白。TOR訊息路徑不只調控Sap2之轉錄，也影響著其蛋白質的量，這可能是透過一般性胺基酸控制(GAAC)之路徑來達成。DNA微陣列分析找到了除了SAP2以外的其他Rhb1下游標的基因。這些發現使吾人在環境營養源、Rhb1-TOR訊息、與白色念珠菌的致病因子表達中獲得新的視野與想法。

關鍵字

白色念珠菌 ； 致病因子 ； 訊息傳遞 ； 致病性

並列摘要

Candida albicans is one of the most common opportunistic fungal pathogens, causing life-threatening disease in immunocompromised patients. We aimed to clarify the regulatory mechanism of some of its virulence factors. We found that the small GTPase Rhb1 and target of rapamycin (TOR) signaling pathway regulates the expression of several C. albicans virulence factors, including secreted aspartyl protease 2 and low nitrogen-mediated morphogenesis. This dissertation comprises two parts. The first part uses site-directed mutagenesis, green fluorescent protein tagging and rapamycin susceptibility assay to show that membrane localization is crucial for Rhb1 activity. In both human and fission yeast, GTP-bound Rhb1 directly activates the TOR kinase. The results indicated that C. albicans Rhb1 may regulate the Tor1 kinase in an indirect manner and Rhb1 has a genetic interaction with the Tsc2 GTPase-activating protein. Moreover, in the absence of Rhb1, the high amount of Tsc2 repressed the Ras1-dependent signaling and caused a defect in low nitrogen-mediated morphogenesis. Ras1 may also involve in TOR signaling pathway upstream or independent of Rhb1.
We next focused on how Rhb1-TOR pathway and the environmental condition changes regulate the expression of secreted aspartyl protease 2 (Sap2), which is the most highly expressed Sap in vitro, and is an important virulence factor. This study shows that Rhb1 is related to cell growth through the control of SAP2 expression when protein is the major nitrogen source. This process involves various components of the TOR signaling pathway, including Tor1 kinase and its downstream effectors. TOR signaling not only controls SAP2 transcription, but also affects Sap2 protein levels, possibly through general amino acid control (GAAC) pathway. DNA microarray analysis identifies other target genes downstream of Rhb1 in addition to SAP2. These findings provide new insights into nutrients, Rhb1-TOR signaling, and expression of C. albicans virulence factor.

並列關鍵字

Candida albicans ； virulence factor ； signal transduction ； pathogenicity

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