Dalton Piotter

Increasing antifungal resistance and limited classes of antifungal drugs pose severe threats for the treatment of fungal infections caused by Candida species. Candida albicans, the most common fungal pathogen, is a common gut and oral opportunistic pathogen of the human microbiome capable of large chromosomal rearrangements that confer drug resistance. One chromosome rearrangement includes an amplification of the left arm of chromosome 5 (Chr5) in an isochromosome structure which promotes drug resistance due to the additional copies of two genes (ERG11 and TAC1). Our experiments have identified that the centromere of Chr5 (CEN5) is flanked by inverted repeat sequences and the CEN5 sequence can become inverted during isochromosome formation. To determine if the CEN5 inversion is independent of the isochromosome structure, 30 single colonies were isolated from a strain that underwent CEN5 inversion and acquired the isochromosome. Single colonies were plated on YPAD and YPAD with 1 microgram fluconazole, and both were incubated overnight. Small and large colonies were observed on both media after 48 hours. Six colonies of each size, small and large, were isolated from drug and non-drug plates. An additional six large colonies were isolated from YPAD. Genomic DNA was extracted and quantified using Nano-drop and Qubit. PCR analysis was performed on CEN5 to determine the orientation of the centromere followed by whole genome sequencing to confirm the absence of the isochromosome and inversion of CEN5. Additional phenotypic analyses were performed, including growth curve analysis in the presence and absence of the antifungal drug.