Identification of Essential Genes and Fluconazole Susceptibility Genes in by Profiling Transposon Insertions.

Within the budding yeasts, the opportunistic pathogen and other members of the clade have developed virulence traits independently from and To begin exploring the genetic basis of virulence and its innate resistance to antifungals, we launched the transposon from a plasmid and sequenced more than 500,000 different semi-random insertions throughout the genome. With machine learning, we identified 1278 protein-encoding genes (25% of total) that could not tolerate transposon insertions and are likely essential for fitness Interestingly, genes involved in mRNA splicing were less likely to be essential in than their orthologs in , whereas the opposite is true for genes involved in kinetochore function and chromosome segregation. When a pool of insertion mutants was challenged with the first-line antifungal fluconazole, insertions in several known resistance genes (, , , , , , , ) and 15 additional genes (including , , ) became hypersensitive to fluconazole. Insertions in 200 other genes conferred significant resistance to fluconazole, two-thirds of which function in mitochondria and likely down-regulate Pdr1 expression or function. Knockout mutants of and , which consume and generate alpha-ketoglutarate in mitochondria, exhibited increased and decreased resistance to fluconazole through a process that depended on Pdr1. These findings establish the utility of transposon insertion profiling in forward genetic investigations of this important pathogen of humans.