利用基因體方法研究人類病原白色念珠菌致病化因素之功能(I)

Abstract

近年來，人類真菌感染(fungal infections) 的情形有日益增加的趨勢。這其中以白色 念珠菌(Candida albicans) 為最大宗，居美國各式院內感染的第四位；相關的醫療費用估 計為每年10 億美金。雖然臨床上有抗真菌藥物，然而這些藥物普遍有副作用、對新出 現及一些特定的菌株無效、及造成抗藥性流行等問題。因此，發展及引進新的觀念及策 略，由不同的角度及方法來瞭解真菌感染的問題是有必要的。 到目前為止，已有幾個與調控白色念珠菌致病機制有關的因素，例如環境刺激、營養 之取得、抗藥性、及毒性因子(virulence factors)(如形態變化、分泌性水解酵素、性狀轉 換)等。但是對整個致病機制的調控迴路(regulatory network) 及各別因素間彼此的關連 及其協同作用仍有很多不清楚。因此，這個研究的長程目標就是要針對白色念珠菌致病 機制的調控因子的作用及其彼此間的關連與協同作用進行瞭解。 在此研究計劃中，我們所要提出的策略是以分子遺傳及功能性基因組為工具-特別著 重在同源置換(homologous replacement) 及去氧核糖核酸微陣列(DNAmicroarray) 來 分析及探索對環境刺激之感應與反應(如特定元素之取得、寄主抗菌反應)及毒性因子(如 形態變化、分泌性水解酵素等)所誘發的致病化(pathogenesis) 過程中，這些因素的訊號 傳遞途徑及其與致病化整體調控迴路的關係。 此子計劃有三個目標： 第一個是由CaEno1 切入，對Efg1 途徑展開功能性研究。Efg1 在白色念珠菌中同時 調控幾個致病化因素，是一個主要的致病化調控樞紐。CaEno1 則受其調節。CaEno1 本 身是具有多功能的蛋白，最早是以醣解酵素的功能被發現；它不但是白色念珠菌細胞壁 的主要成分，也是白色念珠菌感染寄主時的主要抗原，同時也影響白色念珠菌的致病化 過程，因此它在Efg1 途徑中所扮演的角色就很耐人尋味了。由於目前白色念珠菌有性 世代(sexual cycle)尚未被發現，也缺乏適當的質體(plasmid)，因此基因突變的方法將以 同源置換的方式直接對白色念珠菌基因體進行gene targeting 而達成。 第二個目標是以去氧核糖核酸微陣列為工具，刻劃由前述之特定致病機制因素對白色 念珠菌造成的整體基因組的表現。主要是要瞭解各別因素的訊號傳遞途徑及在致病化過 程中對菌體的影響。 第三個目標則是要揭開這些訊號傳遞途徑彼此間協同調控的關連，尤其是與主要致病 化過程中的樞紐Efg1 途徑的關聯。 此研究之成果，除了可以對真菌致病機制的調控有基礎性的瞭解外，更可在過程中揭 露有潛力作為活性阻斷目標的基因產物或訊息傳遞步驟。若將來能有效的阻斷這些目 標，就有可能利用它們及相關知識去發展新一代的抗真菌方法或藥物。因此，此研究計 劃與「基因體醫學國家型科技計畫」的發展目標一致。

Yeast infections in human have increased significantly in recent years. Among the pathogens, Candida albicans is the most dominant one. It has emerged as the fourth most common cause of nosocomial infections in the United States. The estimated cost for treating the Candida nosocomial infections approaches 1 billion US Dollars per year in the United States. Currently available antifungal drugs have undesirable issues such as side effects, ineffective against new or reemerging fungi, and the emerging of resistance. Therefore, development of a new concept and strategy for anti-fungal effort is in demand. Although several factors associated with C. albicans pathogenesis, such as environmental cues, nutrition availability, drug resistance, and virulence factors (for examples, morphogenesis, extracellular hydrolytic activities, and phenotype switch) have been identified, the overall picture of the global networking and the coordination of those regulatory and signaling pathways contributing to the pathogenesis is still missing. Therefore, the long-term goal of this research is to elucidate the regulatory network and mechanisms of pathogenesis in C. albicans, which will allow us the identification of potential targets for anti-fungal purposes. Here, we propose to employ molecular genetics and functional genomics tools, particularly homologous replacement and DNAmicroarray, to analyze and explore (1): the function of signaling/regulatory pathways contributing to the C. albicans pathogenesis induced by the environment cues, iron availability, host anti-microbial responses, and morphogenesis/virulence factors and (2): their roles in the whole gene network of pathogenesis. There are three objectives in this project. The first objective of this study is to focus on the functional study of Efg1 pathway, known to be the key regulatory factor of several pathogenesis pathways, starting from the CaEno1, a known glycolytic proteins specifically regulated by Efg1 and involved in the pathogenesis process as well as being an important cell wall component and the major antigen of Candida infection. What is its function and role in the Efg1 pathway and Candida pathogenesis? Due to the lack of known sexual cycle and plasmids, mutagenesis to study the function of this gene will rely on the homologous replacement technique. The second one is to construct and employ a microarray platform to study the whole-genome profiling induced by selected pathogenesis factors and the regulatory pathways thereof and their global effects on C. albicans. The third is to unveil the relationship of those regulatory/signaling pathways, especially in connection with the Efg1, a major controlling point of pathogenesis, and to map the global gene network of pathogenesis. In addition to satisfy the need for understanding the basic mechanism of fungal pathogenesis, this research may allow us to define particular gene products or signal transduction pathways that can be used as targets to block the transition from commensal to pathogen or to kill the fungal cells specifically. And in the next stage, they may assist in the design and development of new antifungal drugs and/or new antifungal strategies on the molecular level. This study is highly relevant to the aim of innovative research of the 「National Research Program for Genomic Medicine Program」.