Dynamic genome plasticity during unisexual reproduction in the human fungal pathogen Cryptococcus deneoformans

Abstract

Genome copy number variation occurs during each mitotic and meiotic cycle and it is crucial for organisms to maintain their natural ploidy. Defects in ploidy transitions can lead to chromosome instability, which is a hallmark of cancer. Ploidy in the haploid human fungal pathogen Cryptococcus neoformans is exquisitely orchestrated and ranges from haploid to polyploid during sexual development and under various environmental and host conditions. However, the mechanisms controlling these ploidy transitions are largely unknown. During C. deneoformans (formerly C. neoformans var. neoformans, serotype D) unisexual reproduction, ploidy increases prior to the onset of meiosis, can be independent from cell-cell fusion and nuclear fusion, and likely occurs through an endoreplication pathway. To elucidate the molecular mechanisms underlying this ploidy transition, we identified twenty cell cycle-regulating genes encoding cyclins, cyclin-dependent kinases (CDK), and CDK regulators. We characterized four cyclin genes and two CDK regulator genes that were differentially expressed during unisexual reproduction and contributed to diploidization. To detect ploidy transition events, we generated a ploidy reporter, called NURAT, which can detect copy number increases via double selection for nourseothricin-resistant, uracil-prototrophic cells. Utilizing this ploidy reporter, we showed that ploidy transition from haploid to diploid can be detected during the early phases of unisexual reproduction. Interestingly, selection for the NURAT reporter revealed several instances of segmental aneuploidy of multiple chromosomes, which conferred azole resistance in some isolates. These findings provide further evidence of ploidy plasticity in fungi with significant biological and public health implications. Author SummaryPloidy is an intrinsic fundamental feature of all eukaryotic organisms, and ploidy variation and maintenance are critical to the organism survival and evolution. Fungi exhibit exquisite plasticity in ploidy variation in adaptation to various environmental stresses. For example, the haploid opportunistic human fungal pathogen C. deneoformans can generate diploid blastospores during unisexual reproduction and also forms polyploid titan cells during host infection, however, the mechanisms underlying these ploidy transitions are largely unknown. In this study, we elucidated the genetic regulatory circuitry governing ploidy duplication during C. deneoformans unisexual reproduction through the identification and characterization of cell cycle regulators that are differentially expressed during unisexual reproduction. We showed that four cyclin and two cyclin-dependent kinase regulator genes function in concert to orchestrate ploidy transition during unisexual reproduction. To trace and track ploidy transition events, we also generated a ploidy reporter and revealed the formation of segmental aneuploidy in addition to diploidization, illustrating the diverse mechanisms of genome plasticity in C. deneoformans.