在藥物誘導下分離鑑定白色念珠菌之調控因子

Abstract

白色念珠菌的重要性不只在它們感染的嚴重性；而且在長期或預防性治療的病人身上，容易發展出對azole等藥物有抗性的菌株。很多的生化研究因此著重在各式各樣不同的抗azole類的藥物機制。其中最常見的抗藥機制則是藥物排出幫浦 (drug efflux pumps) 基因 (例如CDR1 或 CDR2) 的表現量增加。 近幾年來，很多研究致力於了解白色念珠菌的多重藥物轉運基因(multi-drug transporter genes) CDR1 的調控機制。然而，對於CDR1 的trans調控因子仍然所知不多。本研究因此著重在分離鑑定CDR1的trans調控因子。利用Ym990348 CDR1的啟動子與lacZ的轉譯區 (open reading frame; ORF) 建構成重組基因來當作篩選系統，且在藥物誘導下進行篩選。共有四個ORF被篩選分離出來，此四個ORF分別被命名為REP3, REP4, REP5, 和 REP6。其中兩個 (REP3, REP6) ORF 具有nucleic acid-binding 結構—C2H2 type 指狀區域功能基 (zinc finger domain)，因此它們可能扮演轉譯調控子 (transcription regulator) 的角色。最後根據藥物敏感性測試 (Etest) 的結果，發現REP3 同型缺陷的突變株 (homozygous mutant) 對azole類藥物的感受性有提高的現象。根據以上結果推論，REP3基因參與調控CDR1基因的表現，並且影響白色念珠菌對藥物的感受性。

Candida albicans derive their importance not only from the severity of their infections but also for their ability to develop resistance against antifungals, such as azoles, in patients undergoing long-term or prophylactic treatment. Extensive biochemical studies have highlighted a significant diversity in the mechanisms conferring resistance to azoles. Up-regulation of drug extrusion pump-encoding genes belonging to the ABC (ATP binding cassette, e.g. CDR1 and CDR2) superfamily represents one of the most prevalent mechanisms of Candida drug resistance. In recent years, much effort has been devoted to understand the regulatory mechanisms of multi-drug transporter genes, such as CDR1, in C. albicans. Nonetheless, little is known about the trans-regulatory factors of CDR1. The study here focuses on isolation and identification of the trans-acting regulatory factors of CDR1. Using Ym00348 CDR1 promoter in-frame fusion with lacZ gene as the screening system, under the presence of drugs, four candidate open reading frames (ORFs) have been isolated. These four ORFs were named REP3, REP4, REP5, and REP6. Two (REP3 and REP6) of them have the C2H2 type zinc finger domain, which is one of the major nucleic acid-binding structures, indicating their potential roles as transcription regulators. According to the results of the Etest, rep3/rep3 homozygous mutant seems to be more susceptible to azoles. It shows that REP3 may have involved in the CDR1 expression and may also affect the drug susceptibility of C. albicans.