Ryan Bracken

Candida albicans is a fungal opportunistic pathogen that exhibits genome-scale changes that result in resistance to antifungal drugs. Isolates evolved in nature and in lab experiments contain regions of chromosomes that have been deleted, amplified, and undergone loss of heterozygosity. Many genes in C. albicans are still uncharacterized and how they influence drug resistance or interact to influence drug resistance is unknown. An evolution experiment previously performed in the lab has shown that there is a gene located in a segment of DNA in chromosome 4 called NCP1 that is being overexpressed because of copy number variation. Along with the discovery of NCP1, there is another gene located in chromosome R called KSR1 that undergoes a loss of heterozygosity. Both of these genomic alterations can elevate antifungal resistance in C. albicans. To study the effect of these mutations in combination, we transformed the mutated KSR1 gene into a strain of Candida albicans that already has an overexpression of the NCP1 gene. DNA sequencing was done to confirm that the transformation was successful. We found that this newly transformed strain exhibited an increased resistance to an antifungal drug compared to the wild type. However, the transformed strain was less resistant to an antifungal drug than a strain containing the chromosome 4 amplification and KSR1 loss of heterozygosity. We hypothesize that another gene in the amplification of chromosome 4 is contributing to this resistance. To further investigate this finding we are using the CRISPR overexpression system in a pool of Candida albicans cells to overexpress each gene in the amplified region of chromosome 4. The goal of this future work is to determine what gene is causing the increase in resistance.