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.
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