Identification of a novel point mutation on cap1 during in vitro evolution
Candida albicans is an opportunistic fungal pathogen that is known to exhibit high genomic plasticity. During adaptation to stressors such as antifungal drug, a wide variety of genomic changes can occur. To investigate these genomic rearrangements at the single-cell level, we introduced an optimized dual-fluorescent reporter system into a drug-sensitive strain and serially passaged the engineered strain in the presence of fluconazole, a common antifungal drug. After about 100 generations, we identified a subpopulation with copy number variation (CNV) of chromosome 3 by flow cytometry. Whole genome sequencing of single colonies showed that this CNV was unstable after the drug was removed, as all colonies had lost the CNV. However, a novel point mutation, Cys446*, was identified on the B allele of cap1 and resulted in a truncated protein. Cap1 is a transcription factor primarily known for its role in oxidative stress response, and phenotyping of isolates with the cap1 point mutation revealed the acquisition of drug resistance to fluconazole. We then introduced the same point mutation into the B allele of the drug-sensitive background strain. We found that this point mutation is the cause of the drug resistance, as all resulting strains had increased resistance identical to that of the evolved isolates. Additionally, this point mutation does not cause loss of function, as deletion of either allele of cap1 did not affect the phenotype relative to the background strain. Together, these results show the identification of a novel point mutation at cap1 B allele during in vitro evolution. While previous studies engineered truncated Cap1 that were previously found in S. cerevisiae, our mutation occurred naturally.