白色念珠菌抗藥基因CDR1 之異位調控因子REP3、REP4 與REP5之遺傳學研究

Abstract

白色念珠菌為人體重要的伺機性致病菌，多變性為此微生物的重要特性。最常用以治療白色念珠菌感染的真菌類藥物為azoles類抗真菌藥物，但是由於此類藥物大量且重複性使用已經造成臨床上抗藥性菌株逐漸產生。大部分研究指出真菌最主要的抗藥機制之一就是將藥物排出，使其無法累積於菌體內。Azoles類抗藥性的產生通常和排藥幫浦CDRs(CDR1和CDR2)過量表現有關。CDRs為ATP-binding cassette(ABC)運輸家族。 先前實驗室於啤酒酵母(Saccharomyces cerevisiae)中利用CDR1 promoter-lacZ表現當作報導系統進行白色念珠菌library screening實驗，目的為篩選出白色念珠菌排藥幫浦CDR1的調控者。此次實驗共篩選出了六個可能的ORFs，之後將其命名為REP1，REP2，REP3，REP4，REP5和REP6。本研究是針對REP3、REP4和REP5基因做近一步的分析，探討這些蛋白質在白色念珠菌的抗藥性機制中所扮演的角色。已知REP3基因會譯解出鋅指狀蛋白，且rep3/rep3同型缺陷突變株對於azoles類藥物(fluconazole、voriconazole和itraconazole)的敏感性具有提高現象。將REP3單套基因重新置入rep3/rep3同型缺陷突變株後，於Etest和agar dilution assay中再次驗證REP3基因的確和白色念珠菌與azoles類藥物的敏感性有關，由實驗結果推測Rep3p可能是CDR1的活化子。到目前為止對於REP4基因所知不多。為了找出REP4基因的功能與和CDR1之間的關係，首先利用同源重組破壞REP4基因，之後將單套REP4基因重新放回rep4/rep4同型缺陷突變株進行基因補救，最後也利用大量表現REP4基因觀察其功能。於Etest中發現，大量表現REP4基因時對於fluconazole、voriconazole和itraconazole的敏感性降低，而在agar dilution assay中發現，大量表現REP4基因時對於miconazole的感受性提高。和野生菌株相比，rep4/rep4 對於藥物感受性與芽管形成皆無明顯影響。由實驗結果得知，Rep4p可能和白色念珠菌的抗藥性有關。文獻報導REP5基因可以活化MUC1/FLO11的表現，雖然其自身功能尚不清楚，於白色念珠菌過量表現REP5基因時對於藥物感受性和芽管形成並沒有太大影響。

Candida albicans is an important opportunistic pathogen of humans. A crucial feature of this microorganism is its versatility. The class of azole antifungals is commonly used to treat C. albicans infections and the extensive and repeated use has led to the development of resistance in clinical isolates. The majority of findings point to the increased levels of active efflux of drugs being a prime mechanism of resistance for azole drugs. Azole drug resistance is often associated with the upregulation of genes encoding efflux pumps such as CDRs (CDR1 and CDR2), belonging to the ATP-binding cassette (ABC) transporter superfamily. Previously, a Candida gemonic DNA library screening has been performed in Saccharomyces cerevisiae for identifying the regulators of drug resistance of C. albicans using CDR1 promoter-lacZ expression as the reporter. Six candidate open reading frames (ORFs), namely REP1, REP2, REP3, REP4, REP5 and REP6 were identified. This study focuses on the REP3, REP4 and REP5. The REP3 gene encoded a zinc finger protein and rep3/rep3 homozygous mutants seemed to be more susceptible to azoles (fluconazole, itraconazole and voriconazole). After rescued the homozygous knockout strain with single copy of wild-type REP3 gene, I found that in both Etest and agar dilution assay, REP3 indeed influenced azoles drug susceptibility of C. albicans. According to these results, Rep3p may be an activator of CDR1. There was no direct report concerning REP4 or its homolog. In order to understand the function of REP4 with regard to CDR1 in C. albicans, REP4 homozygous knockout and overexpression strains were constructed by homology recombination and the homozygous knockout was also rescued with single copy of wild-type REP4 gene. In Etest, overexpression of REP4 seemed to be more resistance to fluconazole、voriconazole and itraconazole whereas in agar dilution, overexpression of REP4 seemed to be more susceptible to miconazole. rep4/rep4 strains showed no significant alteration on drug susceptibility and germ tube formation when compared to the wild type. According to these results, Rep4p may influence the drug susceptibility of C. albicans. Through database search, it was suggested that expression of REP5 could activate the expression of MUC1/FLO11, although the function of REP5 itself remained unclear. In this study, overexpression of REP5 did not significantly affect the drug susceptibility and germ tube formation of C. albicans.