臺灣博碩士論文加值系統

熱帶念珠菌(Candida tropicalis)是一種雙倍體的病原真菌。在念珠菌屬中，熱帶念珠菌極具侵略性且是最常見的非白色念珠菌之一。先前的實驗中發現31.25%對flucytosine (5FC)敏感的臨床菌株具有產生抗藥性子代的能力，並進一步證實所觀察到的5FC抗藥性與帶有特定一群多型性核苷酸(SNP)的FCY2基因(會產生運輸cytosine的蛋白質)處在同質二倍體的狀態(homozygous state)有關。本研究將針對熱帶念珠菌對5FC的抗藥機制做更深入的探討。首先，利用另一個關係較遠的臨床菌株建構FCY2同質二倍體的突變株，在5FC藥物敏感性測試實驗中顯示，只有跟上述帶有同樣一群多型性核苷酸的同質二倍體突變株具有抗此藥物的能力。接著分析對5FC極為敏感的臨床菌株在FCY2基因上的各個多型性核苷酸，發現有三個多型性核苷酸只有在具有抗5FC能力的菌株中才存在，經建構這三個多型性核苷酸的點突變株，顯示G145T的點突變加上FCY2基因處在同質二倍體的狀態(145 T/T)與衍生株的抗藥性有關。這個G145T的點突變會造成無意義突變(nonsense mutation)使得FCY2不會合成具有功能的運輸蛋白產物(purine-cytosine permease)。最後，分析全部具有產生抗藥性子代能力的臨床菌株的基因型，發現大部分的親代菌株都帶有145 T/G這種基因型。除了分析FCY2基因與抗5FC藥物之間的關係，本研究也針對兩隻臨床菌株和它們的衍生株之間所發生的異質性缺失(loss of heterozygosity; LOH)做探討。藉由基因多型性圖譜觀察到多數的LOH涵蓋了大部分染色體的區域，甚至延伸到其端粒，這些LOH可能是由於allelic recombination或者break-induced replication所造成。另外有一個小範圍的LOH則可能是由gene conversion所產生。總結，在FCY2基因中產生的無意義突變伴隨大範圍LOH的發生是造成本實驗中熱帶念珠菌衍生株產生抗5FC的主要原因。

Candida tropicalis, a diploid yeast, has been regarded as one of the most invasive and prevalent species of non-albicans Candida. Previous work has shown that 31.25% (30/96) of flucytosine (5FC) susceptible clinical isolates could generate drug resistant progeny. It has revealed that there was an association between a set of polymorphic nucleotides in FCY2 (encoding a cytosine transporter) and the observed drug resistant phenotype. In this study, the molecular mechanisms of 5FC resistance in these C. tropicalis have been further investigated. First of all, homozygous mutants carrying the same set of SNPs in FCY2 under different genetic background displayed similar levels of resistance to 5FC. Second, nucleotide sequences of the FCY2 alleles in hyper-susceptible strains indicated three SNPs in FCY2 were unique to those resistant strains. Subsequently, a homozygous SNP at position 145 (T/T) in the FCY2 gene was proven to be fully accounted for the 5FC resistance observed in the drug resistant derivatives. Since the G145T mutation resulted in a nonsense mutation, it might lead to the loss of purine-cytosine permease. Finally, two types of nonsense mutations (G145T and G201A) were found in FCY2 of the clinical isolates that had potential to produce 5FC resistant derivatives, where the majority was 145 T/G genotype. Meanwhile, the nature and the extent of loss of heterozygosity (LOH) events have been examined by SNP mapping. The results showed that most of the LOH events covered large chromosomal regions, which even extended to the telomere, suggesting allelic recombination or break-induced replication, whereas one was a localized LOH event, caused by gene conversion. All in all, nonsense mutations in FCY2 coupled with LOH events are responsible for 5FC resistance in those C. tropicalis strains.

Chapter 1. Introduction
1.1 Clinical significance of Candida tropicalis 1
1.2 Mechanisms of action of antifungal agents 2
1.3 Molecular mechanisms of flucytosine resistance in yeasts 3
1.4 The association between loss of heterozygosity and antifungal drug resistance 4
1.5 Previous works 5
1.6 Purpose of this study 7

Chapter 2. Materials and Methods
2.1 Materialsts 8
2.1.1 Strains 8
2.1.2 Plasmids and primers 8
2.1.3 Chemicals and reagents 8
2.1.4 Buffers 9
2.1.5 Medium 9
2.1.6 Equipments 9
2.1.7 Strains and media 10
2.2 Methods 10
2.2.1 DNA methods 11
2.2.2 Polymerase chain reaction (PCR) 11
2.2.3 Transformations 12
2.2.4 Constructions of plasmids 13
2.2.5 Sequence analysis 15
2.2.6 Susceptibility testing 15
2.2.7 Quantitative analysis of the mRNA level by real-time PCR 16
2.2.8 Identification of the boundary of loss of heterozygosity (LOH) events 18

Chapter 3. Results
3.1 Phenotypic analysis of the clinical isolates and their homozygous mutants 19
3.2 Sequence analysis of the 5’ and 3’ untranslated regions of the FCY2 locus in hyper- susceltible strains 20
3.3 Construction of plasmids carrying a single mutation at the FCY2 locus 21
3.4 Construction of G-69T (G145T), T-69G (T145G), G273T (G486T), T273G (T486G), C1518T (C1731T) or T1518C (T1731C) single mutation strain 22
3.5 Phenotypic analysis of the single mutation strains 23
3.6 Contribution of the polymorphic nucelotide in the promoter region of the FCY2 gene 25
3.7 Nucleotide sequence analysis of the FCY2 gene 26
3.8 Genotypic characterization of clinical isolates 27
3.9 Characterization of loss of heterozygosity events in clinical isolate-derivative pairs 28
3.10 Characterization of the loss of heterozygosity boundaries 30

Chapter 4. Discussion
4.1 The redefinition of FCY2 open reading frame in C. tropicalis 32
4.2 Contribution of the FCY2 gene to flucytosine susceptibility in C.tropicalis 33
4.2.1 LOH at the FCY2 locus was associated with 5FC resistance 33
4.2.2 The nonsense mutation in the FCY2 gene, accompanied by LOH, contributed to 5FC resistance in C. tropicali 33
4.2.3 Isolates exhibiting 145 T/G genotype might represent a subgroup associated with the generation of 5FC resistant derivatives 35
4.3 Other possibilities that might involve in flucytosine resistance in C. tropicalis 36
4.4 The cause of differential mRNA levels of FCY2 of yeasts carrying a nonsense mutation 37
4.5 Characterization of the loss of heterozygosity events 38

Chapter 5. Future work
5.1 Evaluation of the mechanism of 5FC resistance in C. tropicalis 41
5.2 Examination of the feature of the boundaries that might be related to recombination event 41

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吳佳真 (2009) 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 Loss of heterozygosity contributes to the resistance to 5-flucytosine in Candida tropicalis and the study of identifying the crucial amino acids in the activation domain of CaNDT80