A Tetraploid Intermediate Precedes Aneuploid Formation in Yeasts Exposed to Fluconazole

Candida albicans, the most prevalent human fungal pathogen, is generally diploid. However, 50% of isolates that are resistant to fluconazole (FLC), the most widely used antifungal, are aneuploid and some aneuploidies can confer FLC resistance. To ask if FLC exposure causes or only selects for aneuploidy, we analyzed diploid strains during exposure to FLC using flow cytometry and epifluorescence microscopy. FLC exposure caused a consistent deviation from normal cell cycle regulation: nuclear and spindle cycles initiated prior to bud emergence, leading to trimeras, three connected cells composed of a mother, daughter, and granddaughter bud. Initially binucleate, trimeras underwent coordinated nuclear division yielding four daughter nuclei, two of which underwent mitotic collapse to form a tetraploid cell with extra spindle components. In subsequent cell cycles, the abnormal number of spindles resulted in unequal DNA segregation and viable aneuploid progeny. The process of aneuploid formation in C. albicans is highly reminiscent of early stages in human tumorigenesis in that aneuploidy arises through a tetraploid intermediate and subsequent unequal DNA segregation driven by multiple spindles coupled with a subsequent selective advantage conferred by at least some aneuploidies during growth under stress. Finally, trimera formation was detected in response to other azole antifungals, in related Candida species, and in an in vivo model for Candida infection, suggesting that aneuploids arise due to azole treatment of several pathogenic yeasts and that this can occur during the infection process. Author Summary Fungal infections are a particularly challenging problem in medicine due to the small number of effective antifungal drugs available. Fluconazole, the most commonly prescribed antifungal, prevents cells from growing but does not kill them, giving the fungal population a window of opportunity to become drug resistant. Candida albicans is the most prevalent fungal pathogen, and many fluconazole-resistant strains of this microbe have been isolated in the clinic. Fluconazole-resistant isolates often contain an abnormal number of chromosomes (a state called aneuploidy), and the additional copies of drug resistance genes on those chromosomes enable the cells to circumvent the drug. How Candida cells acquire abnormal chromosome numbers is a very important medical questionis aneuploidy merely passively selected for, or is it actively induced by the drug treatment? In this study, we found that fluconazole and other related azole antifungals induce abnormal cell cycle progression in which mother and daughter cells fail to separate after chromosome segregation. Following a further growth cycle, these cells form an unusual cell type that we have termed trimerasthree-lobed cells with two nuclei. The aberrant chromosome segregation dynamics in trimeras produce progeny with double the normal number of chromosomes. Unequal chromosome segregation in these progeny leads to an increase in the prevalence of aneuploidy in the population. We postulate that the increase in aneuploidy greatly increases the odds of developing drug resistance.