研究受毒性因子EFG1調控之糖解蛋白及分子幫浦在白色念珠菌致病機制中的功能

Abstract

研究受毒性因子EFG1調控之糖解蛋白及分子幫浦在白色念珠菌致病機制中的功能 近年來伺機型真菌造成的感染病症大量地增加。其中，念珠菌(Candida)是最常見的真菌病原。在美國，念珠菌已升為院內感染的第四常見致病菌。在台灣某教學醫院的念珠菌感染案例更是從1981到2000間增加了16倍。我們的長程目標，就是要瞭解白色念珠菌的致病機制及其調控迴路，尤其是形態變化/毒性因子及抗藥性調控，以尋找合適的抗真菌標的及方法。EFG1是白色念珠菌致病機制的主要轉錄因子，影響許多致病途徑的運作，如形態變化、抗藥性、及環境壓力。我們先前DNA 微陣列結果顯示有7個糖解酵素蛋白及8個分子幫浦在4-Nitroquinoline 1-oxide或miconazole藥物影響下受EFG1所調控。由於糖解蛋白涉及能量轉換及/或作為細胞表面成分，而分子幫浦即為細胞表面蛋白，且其運作涉及能量轉換。而且糖解蛋白enolase也被證實除了是念珠菌細胞表面蛋白外，也涉及抗藥性。因此本計畫的目標即是針對這些受EFG1調控的糖解酵素蛋白及分子幫浦來進行功能研究，以瞭解它們及所屬途徑在整個致病機制調控迴路中的角色，特別是在形態變化/毒性因子與抗藥性調控中的作用、彼此的關係、及與EFG1的交互作用。我們除了以knock-out cassette進行同源置換來建構單基因突變株外，也將建構雙基因或多基因突變株，瞭解這些基因在調控迴路中相對的位置以找到特定基因或調控迴路上的位點。此研究所得資訊能幫助我們鑑定可以成為抗真菌藥物的標的與研發新的抗菌策略。

Functional study of glycolytic proteins and efflux pumps under the control of virulence regulator EFG1 in fungal pathogen Candida albicans In the past two decades, yeast infections have played an important role in nosocomial infections. In the United States the yeast infection ranks as the fourth most common cause of nosocomial bloodstream infection. Among the fungal pathogens, Candida albicans is the most frequently isolated fungal pathogen in humans and has caused morbidity in seriously debilitated and immunocompromised hosts. In Taiwan the prevalence of nosocomial candidemia increased 16-fold from 1981 through 2000 at one teaching hospital in Taipei. EFG1, encoding a transcription factor, plays an important role in pathogenesis through several pathways, including morphological switch, drug resistance, and environmental stress. The long-term goal of the research is to unveil the regulatory gene networks of pathogenesis, and at present stage, those centering at EFG1. Glycolytic proteins are enzymes in energy metabolisms and at least one of them, enolase encoded by ENO1, has been shown to be the components of cell surface and also involved in drug resistance. In contrast, efflux pumps are cell surface proteins of which energy is required for the functions and at least two, MDR1 and CDR1 are involved in drug resistance. Our recent systemic study using DNA microarray has found that seven glycolytic proteins and eight efflux pumps were regulated by EFG1. Since both the glycolytic proteins and efflux pumps are potentially involved in energy utilization and cell surface activities, in this study, we would like to study the functions of these glycolytic proteins and efflux pumps under the control of EFG1 to elucidate their functions and regulations, especially their roles in the morphogenesis/virulence and drug resistance gene networks to understand the mechanism of pathogenesis. Furthermore, to reveal their positions in the gene networks of pathogenesis, we also would like to study the interactions among themselves and to EFG1. Since there is no plasmid for C. albicans, we will construct knock-out cassette to replace the target genes from the genome to create null mutants for genetic study. For mapping the relative positions in the networks or pathways, double or multiple gene mutations will also be introduced. The information obtained from this study will allow us to reveal the connection of those genes to the EFG1 pathways and their contribution to the pathogenesis, especially to the morphogenesis/virulence and drug resistance gene networks. In addition, this research may allow us to define particular gene products or regulatory pathways that can be used as targets to block the commensal-to-pathogen transition or kill the fungal cells specifically, and in the next stage, to design and develop new antifungal drugs and strategies targeting the molecular level.