Genome Integrity in Candida albicans

描述（由申请人提供）：口咽念珠菌病通常是人类免疫缺陷病毒（HIV）感染的最早可检测的临床表现，也是获得性免疫缺陷综合征（AIDS）患者中观察到的最普遍的机会性感染。C.白色念珠菌和其它念珠菌属是口咽念珠菌病和食管念珠菌病的最常见原因。鉴于临床上可用的抗真菌药物数量有限，抗真菌药物的耐药性特别令人担忧。这种担忧对于不断增长的免疫功能低下人群尤其重要，特别是在第三世界，他们接受了延长的预防性抗真菌治疗。在上一个资助周期中，我们发现当细胞暴露于唑类抗真菌剂时，非整倍性以高频率获得，并且一种特定的非整倍性，即等染色体5L，通过增加染色体5左臂上基因的拷贝数而引起唑类抗性。从那时起，在其他人类真菌病原体中也发现了非整倍体。这里提出的重要的新数据表明，非整倍体在对最新一类抗真菌药棘白菌素耐药的菌株中很普遍。这个竞争性的更新申请建议继续我们在C.白念珠菌，特别强调基因组如何应对抗真菌药物压力：它如何增加重组水平和染色体拷贝数的变化。我们的长期目标是了解致病真菌用于进化的机制，特别是对抗真菌药物的反应，以便开发干扰这些机制的新治疗方法。使细胞在抗真菌药物存在下存活的过程是伴随疗法的潜在靶点，这将延长有限的可用抗真菌药物库的寿命。一旦我们开发了适当的工具，我们将询问自然对抗真菌药物更具耐药性的非白色念珠菌是否使用类似的抗真菌药物耐药机制。我们将检验由我们以前的工作提出的几个工作假设：a）非整倍性和/或洛是病原真菌用来科普来自使用不同作用机制的不同类别的抗真菌剂的攻击的常见机制; B）特定的细胞亚群获得高水平的非整倍性和/或超重组发生性;和c）四倍体中间体响应于药物胁迫而形成，然后经历协同染色体丢失和/或协同重组事件以产生不同的后代，其中一些后代在胁迫条件下能够更好地存活。我们已经开发了一套强大的工具来分析C中出现的基因组变化的速率和类型。白色念珠菌在这里，我们将使用它们来识别1）与棘白菌素抗性相关的基因组变化类型，2）导致抗真菌药物引起的超重组和非整倍体的机制，以及3）四倍体在抗真菌药物反应中的作用。 公共卫生相关性：真菌感染在免疫功能低下的患者中引起严重的机会性感染，例如感染人类免疫缺陷病毒和获得性免疫缺陷综合征（AIDS）的患者。鉴于临床上可用的抗真菌药物数量有限，抗真菌药物的耐药性特别令人担忧。拟议的工作将解决有关如何应对一系列抗真菌药物产生耐药性的基本问题，目标是确定伴随药物的潜在目标，这些药物将延长有限的抗真菌药物库的寿命。

DESCRIPTION (provided by applicant): Oropharyngeal candidiasis is often the earliest detectable clinical manifestation of infection with Human Immunodeficiency Virus (HIV) and the most prevalent opportunistic infection observed in patients with Acquired Immunodeficiency Syndrome (AIDS). C. albicans and other Candida species are the most frequent causes of oropharyngeal candidiasis and esophageal candidiasis. Resistance to antifungal drugs is of particular concern given the limited number of clinically useful antifungals. This concern is especially critical for the growing population of immunocompromised individuals, especially in the third world, who receive extended courses of prophylactic antifungal therapy. In the previous funding cycle we made the major discovery that aneuploidy is acquired at high frequency when cells are exposed to azole antifungals and that a specific aneuploidy, isochromosome 5L, causes azole resistance by increasing the copy number of genes on the left arm of Chromosome 5. Since then, aneuploidy has been found in other human fungal pathogens. Important new data presented here indicates that aneuploidy is prevalent in strains resistant to the newest class of antifungals, the echinocandins. This competing renewal application proposes to continue our work on genome integrity in C. albicans, with a particular emphasis on how the genome responds to antifungal drug stress: how it increases levels of recombination and undergoes changes in chromosome copy number. Our long-term goal is to understand the mechanisms that pathogenic fungi use to evolve, especially in response to antifungals, so that new therapeutic approaches that interfere with those mechanisms can be developed. The processes that enable cells to survive in the presence of antifungals are potential targets of companion therapies that would extend the life span of the limited arsenal of available antifungals. Once we have develop the appropriate tools, we will ask if non-albicans Candida species, which are naturally more resistant to antifungal drugs, use a similar repertoire of mechanisms of antifungal drug resistance. We will test several working hypotheses raised by our previous work: a) Aneuploidy and/or LOH are common mechanisms used by pathogenic fungi to cope with assault from distinct classes of antifungal agents that use different mechanisms of action; b) Specific sub- populations of cells acquire high levels of aneuploidy and/or are hyper-recombinogenic; and c) Tetraploid intermediates form in response to drug stress and then undergo concerted chromosome loss and/or concerted recombination events to generate diverse progeny, some of which are better able to survive under stress conditions. We have developed a powerful set of tools to analyze the rates and types of genome changes that arise in C. albicans. Here we will use them to identify 1) the types of genome changes associated with echinocandin resistance, 2) the mechanisms that result in hyper-recombination and aneuploidy that occur in response to antifungals and 3) the role of tetraploidy in the response to antifungal drugs. PUBLIC HEALTH RELEVANCE: Fungal infections cause serious opportunistic infections in immunocompromised patients, such as those infected with the Human Immunodeficiency Virus and with Acquired Immunodeficiency Syndrome (AIDS). Resistance to antifungal drugs is of particular concern given the limited number of clinically useful antifungals. The proposed work will address basic questions about how resistance arises in response to a range of antifungal drugs, with the goal of identifying potential targets for companion drugs that would extend the life span of the limited arsenal of available antifungals.