| Summary: | 博士 === 國防醫學院 === 生命科學研究所 === 93 === Candida albicans is an opportunistic pathogen and is the most frequently isolated fungal pathogen in humans. It has caused morbidity and mortality in seriously debilitated and immunocompromised hosts. Increasing reports of clinical fluconazole resistance in C. albicans highlights the need for the examination of the molecular mechanisms responsible for the development of drug resistance. Even though overexpression of efflux pump genes (CDRs, MDR1) are one of the major mechanisms contributing the multidrug resistance in C. albicans, the molecular mechanisms controlling its expression are poorly understood. The goal of this study is to identify the trans-regulatory factors of MDR1 and CDR1. In this study, S. cerevisiae is used as a model to identify the trans-regulatory factors of the efflux pumps. A total of 37 activator and 22 repressor candidates of MDR1 were obtained for further characterization in the future. Two regulatory factor candidates of CDR1 (p4 and p61) were previously identified in Dr. Yun-Liang Yang’s laboratory for increasing the b-galactosidase activity of CDR1p-lacZ in S. cerevisiae. The p4 containing the orf6.8773 with unknown function was named REP1 (Regulator of Efflux Pump). The REP1 gene encoded a putative transcription factor with 693 a.a. in length. It’s DNA binding domain had homologous sequence with that of Ndt80, a transcription factor specifically involved in meiosis in S. cerevisiae. Interestingly, overexpression of REP1 increases susceptibility to fluconazole in S. cerevisiae. Mutations on REP1 increase the susceptibility to fluconazole in C. albicans. The phenotype of increasing susceptibility to antifungals of rep1/rep1 mutant suggests that Rep1 may be an activator of efflux pumps. The other regulatory factor candidate of CDR1﹘p61, containing the ORF of CaNDT80, a putative transcriptional factor with 592 a.a. in length and shared homologous sequence of the DNA binding domain of Ndt80. Overexpression of CaNDT80 decreases susceptibility to fluconazole and ketoconazole in S. cerevisiae. In consistency, the susceptibilities to azoles and flucytosine of the CaNDT80 mutant cells are increased. Mutations on CaNDT80 reduce the expression of CDR1 to 15% and abolish the induction of CDR1 expression by antifungal agents in C. albicans. In addition to drug susceptibility, mutations on CaNDT80 also result in cells that trap in a pseudohyphal growth and fail to develop true hyphae. Consequently, mutations on CaNDT80 also reduce the virulence in C. albicans in a mouse model. In the present study, it has been shown that both virulence and drug resistance pathways of C. albicans can be co-regulated by one key protein, CaNdt80. Among the regulatory factors, CaNDT80 may be the first gene whose function has been identified to be involved in both drug resistance and virulence in C. albicans. Hence, the findings in this study may open a new concept for developing and designing new effective agents for microbial infections.
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