Centromere Structure and Function in Candida albicans

描述(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命性全身感染。预防性抗真菌治疗往往会导致获得性耐药的出现。白念珠菌不经历减数分裂；遗传多样性是通过体细胞有丝分裂事件而产生的，例如染色体不分离。我们最近发现，对氟康唑的获得性耐药通常是通过涉及5号染色体着丝粒的基因组重排来实现的。着丝粒(CENS)是蛋白质聚集形成动点的DNA区域，动点是将染色体连接到有丝分裂纺锤体的结构。它们对适当的染色体分离以及最终对基因组稳定性和细胞存活至关重要。白色念珠菌的区域中心与包括人类在内的高等生物的区域中心相似，也不同于酿酒酵母特征明确的点着丝粒。重要的是，白色念珠菌比最小的区域性致病菌要小得多，也更简单，因此为利用白色念珠菌作为区域致病菌研究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，新着丝粒是在非着丝粒DNA上形成的异位着丝粒。此外，我们还构建了可用于多种应用的CEN-质粒。我们的长期目标是：a)了解白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，促进对白念珠菌基本和应用过程的研究。具体地说，我们将开发使用ADE2标记的CEN-质粒的菌株，以有效地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疗法至关重要：一种是通过直接靶向不同于人类着丝粒的基本真菌着丝粒成分来杀死真菌的药物；另一种是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，以扩大其有效性。我们建议：1)确定CEN在天然染色体中的功能要求；2)确定CEN功能在CEN-质粒上建立和维持的要求；3)使用CEN-质粒筛选影响染色体稳定性和耐药性获得的候选抗真菌药物和伴随药物，并可能延长现有抗真菌药物的有效性。 公共卫生相关性：真菌感染是一个严重的健康问题，这是由于可用的抗真菌药物数量有限，以及临床上看到的抗真菌药物迅速产生抗药性。白色念珠菌是人类最常见的真菌病原体，其获得性耐药源于染色体分离缺陷。我们将开发工具来研究这一过程，并将确定抑制这些缺陷的配套药物，从而扩大现有抗真菌疗法的有效性。

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.