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Within the budding yeasts, the opportunistic pathogenCandida glabrataand other members of theNakaseomycesclade have developed virulence traits independently from the CTG clade that includesCandida albicans. To begin exploring the genetic basis ofC. glabratavirulence and its innate resistance to antifungals, we launched theHermestransposon from a plasmid and obtained more than 500,000 different semi-random insertions throughout the genome. Using machine learning, we identify up to 1278 protein-encoding genes (25% of total) that cannot tolerate transposon insertions and are thus essential forC. glabratafitness in vitro. Interestingly, genes involved in mRNA splicing were less likely to be essential inC. glabratathan their orthologs inS. cerevisiae, whereas the opposite is true for genes involved in kinetochore function and chromosome segregation. Insertions in several known genes (e.g.PDR1,CDR1,PDR16,PDR17,UPC2A,DAP1) caused hypersensitivity to the first-line antifungal fluconazole, and we identify 12 additional genes that also contribute to innate fluconazole resistance (KGD1,KGD2,YHR045W, etc). 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. These findings show the utility of transposon insertion profiling in genome-wide forward-genetic investigations of fungal pathogens.IMPORTANCEPathogenic yeasts cause mucosal and systemic infections in millions of people each year. The innate resistance ofCandida glabratato fluconazole and its ability to acquire resistance to 2 other antifungals are contributing to its rise in incidence. Our understanding ofC. glabratabiology has been hampered by inefficient genetic and genomic tools. This study addresses those deficiencies by developing powerful transposon mutagenesis strategies for the first time in this pathogen. We identify nearly all essential genes ofC. glabratathat could be targeted for development of new antifungals. We generate large pools of random insertion mutants that can be easily monitoreden massewith deep sequencing, thus enabling identification of genes involved in any number of biological processes. We identify dozens of new genes that increase or decrease innate resistance of clinical isolate BG2 to fluconazole and provide resources for further exploration ofC. glabratagenetics and genomics. |
