Candida albicans is an important human fungal pathogen that causes superficial mucosal and lethal systemic infections. Prophylactic antifungal treatments often result in the appearance of acquired drug resistance. C. albicans does not undergo meiosis;genetic diversity arises through somatic mitotic events such as chromosome non-disjunction. We recently found that acquired resistance to fluconazole is often conferred by genome rearrangements involving the centromere of chromosome 5. Centromeres (CENs) are the DNA regions where proteins assemble to form kinetochores, the structures that tether chromosomes to the mitotic spindle. They are critical to proper chromosome segregation and ultimately to genome stability and cell survival. C. albicans has regional CENs like those of higher organisms including humans, and unlike the well characterized point centromeres of Saccharomyces cerevisiae. Importantly, C. albicans CENs are much smaller and simpler than the smallest characterized regional CENs, thus providing a unique opportunity to use C. albicans as a model for the study of regional CENs. Our preliminary results show that deletion of a CEN region can be accompanied by formation of a neocentromere, an ectopic centromere that forms at non- centromeric DNA. Furthermore, we have constructed CEN-plasmids that will be useful for many applications. Our long term goals are: a) to understand the DNA and proteins that specify C. albicans centromere function;b) to design useful molecular tools that exploit our understanding of centromere function and facilitate the study of basic and applied processes in C. albicans. Specifically, we will develop strains that use ADE2-marked CEN-plasmids in a powerful screen for compounds with potential therapeutic value. Our work is critical for the development of two types of potential therapies directed at centromeres: drugs that kill fungi by directly targeting essential fungal centromere components that differ from those in the human centromere;and companion therapies that eliminate aneuploidies and therefore could be administered together with currently available antifungals in order to extend their usefulness. We propose to: 1) determine the requirements for CEN function within native chromosomes;2) determine the requirements for the establishment and maintenance of CEN function on CEN-plasmids;and 3) use CEN-plasmids to screen for candidate antifungal drugs and companion drugs that affect chromosome stability and the acquisition of drug resistance and could extend the usefulness of existing antifungal drugs.
Fungal infections are a serious health problem due to the limited number of antifungal drugs available and the rapid acquisition of resistance to antifungals seen in the clinic. In Candida albicans, the most prevalent fungal pathogen of humans, acquired drug resistance arises from defects in chromosome segregation. We will develop tools to study this process and will identify companion drugs that inhibit these defects, thereby extending the usefulness of available antifungal therapies.
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