| Summary: | 碩士 === 國立交通大學 === 生化工程研究所 === 95 === Candida albicans is an opportunistic fungal pathogen and is the most common cause of deep mycoses in humans. Azole therapy is commonly used to treat C. albicans infections. However the widespread use of azoles led to an increased frequency of treatment failure due to azole-resistant C. albicans in clinical setting. Therefore, understanding the molecular mechanisms of drug resistance in C. albicans is important to render the fungal infection. Over expression of drug efflux pumps— CDR1 and CDR2 (Candida Drug Resistance 1 and 2) in azole resistant C. albicans is commonly observed but the regulatory mechanism is poorly understood. Previously in the laboratory, REP3 (Regulator of Efflux Pump) was isolated from Candida genomic library due to its ability to increase the ��-galactosidase activity of CDR1YM990348 promoter-lacZ about four folds in Saccharomyces cerevisiae in the presence of miconazole. According to the results of the drug susceptibility tests, rep3/rep3 homozygous mutant seems to be more susceptible to miconazole, itraconazole, ketoconazole, fiuconazole and voriconazole than the SC5314 wild-type strain in spite of rep3/rep3::REP3 rescued strains not showing a restoration of drug susceptibility phenotype. In this study, REP3 rescued strains were re-constructed and the outcome remained the same. No REP3 mRNA could be detected in rep3/rep3 mutant and the real-time PCR results showed that the REP3 mRNA could be detected in the rep3/rep3::REP3 rescued strains in similar quantity as that of the SC5314 wild-type strain. Surprisingly, the expressions of CDR1 were similar in the rep3/rep3 mutants, rep3/rep3::REP3 rescued strains, and SC5314 wild type strain. Furthermore, the expression level of the orf19.3926, which locates at the down stream 266 bps from REP3, increased about 3.5 folds in the REP3 rescued strain than that of the wild-type strains and rep3/rep3 mutants in the presence of miconazole. Even though the REP3 rescued strains were reconstructed through SAT1 flipping method to eliminate the potential interference from the integration of vector DNA fragment in C. albicans, the regenerated rep3/rep3::REP3 rescued strains still cannot restore completely the drug susceptibility phenotype of rep3/rep3 mutant. Since the mRNA level of CDR1 in rep3/rep3 mutant was the same as that in the wild type. REP3 may be involved in drug resistance through pathways other than CDR1 in C. albicans. There is no clear reason as the why the rep3/rep3::REP3 rescued strain could not restore the REP3 phenotype. Both will require further studies.
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