Centromere Structure and Function in Candida albicans

描述（由申请人提供）：白色念珠菌是一种重要的人类真菌病原体，可引起浅表粘膜和致死性全身感染。预防性抗真菌治疗往往导致获得性耐药性的出现。白色念珠菌不经历减数分裂；遗传多样性产生于体细胞有丝分裂事件，如染色体不分离。我们最近发现，对氟康唑的获得性耐药性通常是由涉及5号染色体着丝粒的基因组重排引起的。着丝粒（CENs）是蛋白质聚集形成着丝点的DNA区域，着丝点是将染色体连接到有丝分裂纺锤体的结构。它们对正确的染色体分离至关重要，最终对基因组稳定和细胞存活至关重要。白色念珠菌与包括人类在内的高等生物一样具有区域性ccn，而不像酿酒酵母那样具有充分表征的点着丝粒。重要的是，白色念珠菌cns比最小的特征区域cns更小，更简单，从而为使用白色念珠菌作为研究区域cns的模型提供了独特的机会。我们的初步结果表明，CEN区域的缺失可能伴随着新着丝粒的形成，这是一种在非着丝粒DNA上形成的异位着丝粒。此外，我们已经构建了可用于许多应用的cen质粒。我们的长期目标是：a)了解指定白色念珠菌着丝粒功能的DNA和蛋白质；b)设计有用的分子工具，利用我们对着丝粒功能的理解，促进白色念珠菌基础和应用过程的研究。具体来说，我们将开发使用ade2标记的cen质粒的菌株，用于筛选具有潜在治疗价值的化合物。我们的工作对于开发两种针对着丝粒的潜在治疗方法至关重要：通过直接靶向与人类着丝粒不同的重要真菌着丝粒成分来杀死真菌的药物；消除非整倍体的伴随疗法因此可以与目前可用的抗真菌药物一起使用以延长其效用。我们建议：1)确定原生染色体对CEN功能的要求；2)确定CEN质粒建立和维持CEN功能的要求；3)利用cen -质粒筛选影响染色体稳定性和获得耐药性的候选抗真菌药物和伴随药物，从而扩大现有抗真菌药物的使用范围。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: 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.