分離鑑定白色念珠菌CDR1基因之調控因子

Abstract

中文摘要 許多目前使用治療真菌的藥物皆具有副作用、對某些真菌治療效果不佳及抗藥性產生等問題。其中azole-based藥物相對副作用較小且使用方便，因此成為臨床上重要治療真菌的藥物。但由於使用頻繁，抗藥性問題也日益嚴重。Azole-based 藥物主要抗藥機制中包括藥物排出幫浦（efflux pumps）基因CDRs （CDR1與CDR2）與MDR1 mRNA表現增加。白色念珠菌CDR1基因所轉譯出的藥物排出幫浦蛋白可將菌體內的藥物排出。為了找出CDR1 表現與哪些trans 調控因子有關，首先構築ADE3-CDR1-LacZ in-frame fusion integrative plasmid，接著利用homologous recombination，將此質體插入Saccharomyces cerevisiae ADE3基因位置，此菌株編號為YSHI 10，利用YSHI 10與白色念珠菌基因庫來篩選trans 調控因子。在濾紙上分析 b-galactosidase ，挑出5株深藍色菌落P4、P7、P11、P34與P61做進一步研究。發現P 4與P 61菌株其b-galactosidase表現與transform入之白色念珠菌之基因庫有關。由P 4與P 61抽出帶有白色念珠菌genomic DNA質體p 4與p 61進行定序與分析，p 4含有2694 bps genomic DNA，是由兩段genomic DNA組成，內含一open reading frame跨越此兩段genomic DNA，而p 61含有5040 bps genomic DNA亦是由兩段genomic DNA組成，含有一個open reading frame跨越此兩段genomic DNA。另外，我也由兩株台灣臨床菌株Ym990348與Ym990361構築CDR1P-LacZ fusion plasmids，以濾紙分析b-galactosidase，我發現兩株臨床菌株CDR1啟動子不具活性，所以將Ym990348、Ym990361與SC5314的CDR1 promoters進行比對，結果發現Ym990348與Ym990361 的CDR1 promoters非常相似，但兩者與SC5314的CDR1 promoters在-600至start codon ATG之間有多處不同，值得進一步探討。

Abstract Many of the current available antifungal drugs have undesirable side effects, and also are sometimes ineffective to against new or reemerging fungi. The rapid development of resistance is great concern. The azole antifungal agents , because of their relative safety and ease of delivery , have subsequently become a critical component in the antifungal armamentarium . The widespread use of azole drugs has led to the development of drug-resistant isolates. Several molecular mechanisms that contribute to drug resistance have been identified, increasing mRNA levels for two types of efflex pump genes : the ATP binding cassette transporter CDRs（CDR1 and CDR2）and the major facilitator MDR1. The CDR1 gene encodes a multidrug transporter in Candida albicans . In order to isolate and identify the trans-regulatory factors of CDR1 in Candida albicans, the ADE3-CDR1P-LacZ in frame fusion integrative plasmid has been constructed . Because of the homologous recombination, the plasmid integrated into the ADE3 gene in Saccharomyces cerevisiae . The strain was named YSHI 10. YSHI 10 was used to screen Candida albicans, genomic library. Acorrding to the b-galatosidase assay, P 4 and P 61 had higher level b-galatosidase expression, which is associated to the possible activator within Candida albicans, genomic DNA. I further extracted the plasmid DNA from P 4 and P 61 and sequence the DNA fragments. Where was the plasmid from the P 4 strains contained about 2694 bps of Candida albicans, genomic DNA with one open reading frame. P 61 strains contained about 5040 bps of Candida albicans, genomic DNA and contained one open reading frame. In addtion , I also constructed two CDR1P-LacZ fusion plasmids from clinical isolates of Candida albicans Ym990348 and Ym990361 in Taiwan . In filter b-galatosidase assay, It was found that both of the CDR1P in Ym990348 and Ym990361 had no expression. Then I compared the three CDR1P sequences in Ym990348, Ym990361 and SC5314. Finding CDR1P sequences in Ym990348 and Ym990361 are very similar. But there are several different from CDR1P in SC5314 between –600 to start coden ATG which worthes further study. ii