Functional genomic analysis of genes important for Candida albicans fitness in diverse environmental conditions

As the limited arsenal of effective antifungals in clinical use is threatened by emerging resistance, the demand for new strategies to combat invasive fungi is urgent. One strategy to expand the therapeutic target space is to identify genes important for pathogen growth in host-relevant environments. Here, we leveraged a pooled functional genomic screening strategy to identify and characterize genes important for fitness of the human fungal pathogen Candida albicans in diverse conditions. Through this approach we identified a novel essential gene with no known Saccharomyces cerevisiae homolog, C1_09670C, and demonstrated that it encodes subunit 3 of replication factor A, Rfa3. Furthermore, we applied robust computational analyses to leverage profile similarity analysis of mutant phenotypes across conditions to identify functionally-coherent gene clusters and predict gene function. Through this approach, we correctly predicted the cell cycle-associated function of C3_06880W, a previously uncharacterized genes required for fitness specifically at elevated temperatures. Protein structure predictions coupled with genetic and biochemical assays confirmed C3_06880W encodes Iml3, a component of the C. albicans kinetochore with key roles in C. albicans virulence in vivo. Overall, this work reveals novel insights into the vulnerabilities of C. albicans. To enable systematic fitness comparisons between diverse growth conditions, we utilized a pooled functional genomics approach for massively parallel analysis of Candida albicans GRACE strains to assess each strain’s fitness profile through quantification of strain-specific barcodes by next-generation sequencing. Specifically, a pooled collection of 2,238 barcoded GRACE strains covering ~35% of the genome was grown in triplicate in the presence and absence of DOX for 24 hours, then sub-cultured into matching fresh medium with or without DOX for an additional 48-hour incubation. ). Screens in all conditions were performed in technical triplicate and biological duplicate apart from the baseline condition (YNB at 30 ˚C), which was tested in five biological replicates. After growth of the cultures, strain-specific molecular barcodes were amplified from extracted gDNA, and pooled barcode PCR products were subjected to next-generation sequencing.