熱帶念珠菌失去異質合子性導致5-Flucytosine抗藥性及鑑別CaNDT80活化區重要胺基酸之研究

Abstract

實驗室先前在 C. tropicalis 臨床菌株的 5-flucytosine (5-FC) 感受性測試中分離到抗藥衍生株。根據前人研究，破壞 pyrimidine 生合成途徑任何一個蛋白質，或是影響其調控都會導致 5-FC 抗藥性。於是將 parental strain 與其抗藥衍生株進行相關抗藥基因 FCY1、FCY2、FUR1 和 URA3 序列分析，發現有些 parental strains 的 FCY2 為異質合子型，但其抗藥衍生株卻變成同質合子型。因此更進一步以 SAT1 flipper 建構 FCY2 同質合子置換株及單股缺陷突變株，並證實失去異質合子性與 5-FC 抗藥性發生有關。但 FCY2 失去異質合子性所造成的 5-FC 抗藥有兩個可能原因，第一， Fcy2p 第 91 個胺基酸由 Met 變為 Ile；第二，promoter 序列的變異使 FCY2 mRNA 表現量下降。 本研究第二部分與排藥幫浦 CDR1 相關，文獻指出其大量表現與 azoles 抗藥性相關，然而其調控機制目前瞭解並不多。實驗室先前於 C. albicans genomic DNA library 中篩選到 CaNDT80 為 CDR1 可能的正向轉錄因子。經由序列比對CaNDT80 與 S. cerevisiae 的 ScNDT80 為同源基因，它們的DNA結合區有 35％ 相同度和 53％ 相似度，有趣的是活化區卻沒有任何相似性，這是一段全新的序列，沒有人研究過它的功能。因此利用 error-prone PCR 在 CaNDT80 活化區產生隨機突變，建構成一個 library，想要找出哪些胺基酸對於調控 CDR1 扮演重要的角色。為了達到這個目標，於是分別建構了以 chloramphenicol 及 kanamycin 為報導基因的篩選性質體的 clones，用來剔除 library 中帶有 nonsense 及 frameshift mutations 的 clones，以利日後的研究。初步測試顯示於適當濃度的抗生素培養基中，此二質體均具有篩選功能，可作為之後的應用。

In the laboratory, C. tropicalis clinical isolates were selected for derivatives resistant to 5-flucytosine (5-FC). Researchers have previously shown that disruption of the function of any protein involved in pyrimidine biosynthetic pathway or regulation could contribute to 5-FC resistance. Accordingly, four genes , FCY1, FCY2, FUR1 and URA3 involved in pyrimidine biosynthetic pathway was sequenced. The result revealed that some strains were heterozygous in FCY2 encoding purine cytosine permease (PCP), whereas their drug-resistant derived strains were homozygous. I further used the SAT1 flipper to generate homozygous strains and single allele mutants in FCY2 and demonstrated that the 5-FC resistance was associated with loss of heterozygosity (LOH) in C. tropicalis. According to the result, there are two possibilities of LOH in FCY2 contributing to 5-FC resistance. One was that the change of M91I in PCP was affecting the function and the other was that FCY2 mRNA expression was decreased in the resistant allele. My second project was to study the regulation of drug resistance. Previously in the laboratory, a C. albicans genomic DNA library screening identified CaNDT80 as a positive regulator of CDR1, an efflux pump for the azole resistance in C. albicans. CaNdt80p is 672 amino acids in length and homologous to ScNdt80p (592 aa) in S. cerevisiae. There are 35% identity and 53% similarity between their DNA-binding domains. Interestingly, their activation domains share no similarity. The sequence of the activation domain of CaNdt80p is novel and has not yet been characterized. Hence, I have conducted a study to construct the library containing randomly mutated DNA sequences of the activation domain of CaNDT80 by error-prone PCR to identify important residues for the function and also constructed two plasmids using chloramphenicol and kanamycin, separately, as reporters to eliminate the undesired nonsense and frameshift mutations in the library for future studies. According to testing results, reporter plasmids are potential tools with selective functions in appropriate conditions.