Genome Integrity in Candida albicans

描述(申请人提供)：机会性感染仍然是感染人类免疫缺陷病毒(HIV)的最重要的并发症，也是获得性免疫缺陷综合症(AIDS)患者死亡的主要原因。这些患者中有很大一部分发展为严重的口咽和食道念珠菌病，使用唑类抗真菌药物如氟康唑治疗，导致耐唑类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(非整倍体)的变化和与耐药性有关的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它适应人类宿主中遇到的压力，如果是的话，确定它这样做的分子机制。我们将使用三种强大的工具来分析整个白念珠菌基因组的遗传变化：单核苷酸多态(SNP)阵列将监测整个白念珠菌基因组中的杂合性丢失事件；比较基因组杂交阵列将检测整个染色体或染色体片段(非整倍体)的丢失或获得；反选择GAL1基因将被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的基因类型，比较基因组杂交阵列准确地检测到染色体拷贝数的增加和减少，当细胞在体内繁殖时，白色念珠菌细胞中的重组频率增加，并且杂合性和/或非整倍体的丧失发生在表现出更高的耐药性水平或增加的寄主能力的菌株中。在这项提议中，我们将检验这样的假设，即白色念珠菌对其在宿主中遇到的压力和/或当暴露于非整倍体水平增加和/或杂合性丧失的抗真菌药物时做出反应。此外，我们将询问在这些压力条件下，特定的基因组变化是否为白色念珠菌提供了选择优势。我们将检测氟康唑耐药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、比率和类型，并将确定特定的基因组片段和氟康唑耐药所必需的基因。我们提出的研究有可能做出两个独特的贡献。可能会发现具有更高耐药潜力的特定病原体基因类型，可能会发现通过使用配套药物或疗法来保存现有抗真菌药物疗效的改进策略。

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.