Genome Integrity in Candida albicans

描述(申请人提供)：口咽念珠菌病通常是感染人类免疫缺陷病毒(HIV)的最早可检测到的临床表现，也是在获得性免疫缺陷综合征(AIDS)患者中观察到的最常见的机会性感染。白念珠菌和其他念珠菌是口咽念珠菌病和食道念珠菌病最常见的原因。鉴于临床上使用的抗真菌药物数量有限，对抗真菌药物的耐药性尤其令人担忧。这一担忧对于不断增长的免疫受损个体尤其重要，尤其是在第三世界，他们接受了长期的预防性抗真菌治疗。在前一个资助周期中，我们的主要发现是，当细胞暴露在唑类抗真菌药物中时，非整倍体的获得频率很高，并且一种特殊的非整倍体，即等染色体5L，通过增加5号染色体左臂上的基因拷贝数而导致唑类耐药性。此后，在其他人类真菌病原体中也发现了非整倍体。这里提供的重要新数据表明，非整倍体在对最新一类抗真菌药物棘球菌素类药物耐药的菌株中普遍存在。这一竞争性的更新应用程序建议继续我们在白念珠菌基因组完整性方面的工作，特别强调基因组如何对抗真菌药物压力做出反应：它如何增加重组水平和经历染色体拷贝数的变化。我们的长期目标是了解病原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的治疗方法。使细胞在抗真菌药物存在的情况下存活的过程是伴随疗法的潜在目标，这些疗法将延长有限的现有抗真菌药物武器库的寿命。一旦我们开发出适当的工具，我们就会问，自然对抗真菌药物更具抗药性的非白念珠菌物种是否使用了类似的抗真菌耐药性机制。我们将检验我们以前的工作提出的几个工作假说：a)非整倍体和/或杂合性缺失是病原真菌用来应对不同类型抗真菌药物攻击的常见机制，这些机制使用不同的作用机制；b)特定的细胞亚群获得高水平的非整倍体和/或超重组；以及c)四倍体中间体对药物胁迫做出反应，然后经历染色体协调丢失和/或协调重组事件，以产生不同的后代，其中一些在应激条件下能够更好地生存。我们已经开发了一套强大的工具来分析出现在白色念珠菌中的基因组变化的速度和类型。在这里，我们将使用它们来鉴定1)与棘球绦虫耐药相关的基因组变化的类型，2)导致抗真菌药物反应中发生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。

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.