Genome Integrity in Candida albicans

描述（由申请人提供）：寄生虫感染仍然是人类免疫缺陷病毒（HIV）感染的最重要并发症，也是获得性免疫缺陷综合征（AIDS）患者死亡的主要原因。这些患者中的很大一部分发展为严重的口咽和食管念珠菌病，并且用唑类抗真菌药物如氟康唑治疗，导致唑耐药念珠菌菌株的患病率增加。许多耐药菌株表现出遗传改变，包括染色体拷贝数的变化（非整倍性）和耐药重要基因的杂合性丢失。我们的长期目标是了解白色念珠菌基因组的不稳定性是否有助于其适应人类宿主中遇到的压力，如果是，则确定其分子机制。我们将使用三个强大的工具来分析整个C的遗传变化。将使用白色念珠菌基因组：单核苷酸多态性（SNP）阵列将监测整个念珠菌杂合性丢失事件。白色念珠菌基因组;比较基因组杂交阵列将检测整个染色体或染色体片段的丢失或获得（非整倍体）;并且反向选择GAL 1基因将用于分离体内和体外罕见的杂合性丢失事件。初步结果表明，SNP阵列很容易区分纯合和杂合基因型，比较基因组杂交阵列准确检测染色体拷贝数的增加和减少，C.当细胞在体内繁殖时，白念珠菌细胞的杂合性和/或非整倍性的缺失在表现出增加的耐药性水平或增加的定殖宿主的能力的菌株中发生。在这个建议中，我们将测试的假设，C。白色念珠菌对它在宿主中遇到的压力和/或当暴露于抗真菌药物时的反应具有增加的非整倍性水平和/或杂合性丢失。此外，我们将询问特定的基因组变化是否提供了C。白色念珠菌在这些应激条件下具有选择性优势。我们将检测氟康唑耐药菌株的非整倍体和杂合性丢失，测量发生杂合性丢失事件的频率、速率和类型，并将鉴定氟康唑耐药所需的特定基因组片段和基因。我们提出的研究有可能做出两个独特的贡献。可以鉴定出具有更高的耐药性潜力的特定病原体基因型，可以发现通过使用伴随药物或治疗来保持现有抗真菌剂功效的改进策略。

DESCRIPTION (provided by applicant): Opportunistic infections remain the most important complication of infection with the Human Immunodeficiency Virus (HIV) and the principal cause of death in patients with the Acquired Immune Deficiency Syndrome (AIDS). A large proportion of these patients develop severe oropharyngeal and esophageal candidiasis and treatment with the azole class of anti-fungal drugs such as fluconazole, has led to an increase in the prevalence of azole-resistant Candida strains. Many drug-resistant strains exhibit genetic alterations including changes in chromosome copy number (aneuploidy) and loss of heterozygosity at genes important for drug resistance. Our long-term goal is to understand whether Candida albicans genome instability facilitates its adaptation to stresses encountered in the human host and, if it does, to determine the molecular mechanisms by which it does so. We will use three powerful tools to analyze genetic changes across the entire C. albicans genome will be used: Single Nucleotide Polymorphism (SNP) arrays will monitor loss of heterozygosity events throughout the C. albicans genome; Comparative Genome Hybridization arrays will detect the toss or gain of whole chromosomes or chromosome fragments (aneuploidy); And the counter-selectable GAL1 gene will be used to isolate rare loss of heterozygosity events in vivo and in vitro. Preliminary results indicate that the SNP arrays readily distinguish homozygous and heterozygous genotypes, that the Comparative Genome Hybridization arrays accurately detect increases and decreases in chromosome copy number, that recombination frequency in C. albicans cells is elevated when cells are propagated in vivo and that loss of heterozygosity and/or aneuploidy occurs in strains that exhibit increased levels of drug resistance or increased ability to colonize the host. In this proposal, we will test the hypothesis that C. albicans responds to stresses it encounters in the host and/or when exposed to antifungal drugs with increased levels of aneuploidy and/or loss of heterozygosity. Further, we will ask if specific genome changes provide C. albicans a selective advantage under these stress conditions. We will detect aneuploidy and loss of heterozygosity in fluconazole resistant strains, measure the frequency, rates and types of loss of heterozygosity events that occur, and will identify specific genome segments and genes necessary for fluconazole resistance. Our proposed research has the potential to make two unique contributions. Specific pathogen genotypes that have a higher potential to become drug resistant may be identified, improved strategies for preserving the efficacy of existing antifungal agents through the use of companion drugs or therapies may be discovered.