Search for: All records

The genetic background between strains of a single species and within a single strain lineage can significantly impact the expression of biological traits. This genetic variation may also reshape epigenetic mechanisms of cell identity and environmental responses that are controlled by interconnected transcriptional networks and chromatin-modifying enzymes. Histone deacetylases, including sirtuins, are critical regulators of chromatin state and have been directly implicated in governing the phenotypic transition between the ‘sterile’ white state and the mating-competent opaque state inCandida albicans,a common fungal commensal and pathogen of humans. Here, we found that a previously ambiguous role for the sirtuinSIR2inC. albicansphenotypic switching is likely linked to the genetic background of mutant strains produced in the RM lineage of SC5314.SIR2mutants in a specific lineage of BWP17 displayed increased frequencies of switching to the opaque state compared to the wild-type. Loss ofSIR2in other SC5314-derived backgrounds, including newly constructed BWP17sir2Δ/Δ mutants, failed to recapitulate the increased white–opaque switching frequencies observed in the original BWP17sir2Δ/Δ mutant background. Whole-genome sequencing revealed the presence of multiple imbalanced chromosomes and large loss of heterozygosity tracts that likely interact withSIR2to increase phenotypic switching in this BWP17sir2Δ/Δ mutant lineage. These genomic changes are not found in other SC5314-derivedsir2Δ/Δ mutants that do not display increased opaque cell formation. Thus, complex karyotypes can emerge during strain construction that modify mutant phenotypes and highlight the importance of validating strain background when interpreting phenotypes. 

TheCandida albicansgenome contains between ten and fifteen distinctTLOgenes that all encode a Med2 subunit of Mediator. In order to investigate the biological role of Med2/Tlo inC.albicanswe deleted all fourteenTLOgenes using CRISPR-Cas9 mutagenesis. ChIP-seq analysis showed that RNAP II localized to 55% fewer genes in thetloΔ mutant strain compared to the parent, while RNA-seq analysis showed that thetloΔ mutant exhibited differential expression of genes required for carbohydrate metabolism, stress responses, white-opaque switching and filamentous growth. Consequently, thetloΔ mutant grows poorly in glucose- and galactose-containing media, is unable to grow as true hyphae, is more sensitive to oxidative stress and is less virulent in the wax worm infection model. Reintegration of genes representative of the α-, β- and γ-TLOclades resulted in the complementation of the mutant phenotypes, but to different degrees.TLOα1could restore phenotypes and gene expression patterns similar to wild-type and was the strongest activator of glycolytic and Tye7-regulated gene expression. In contrast, the two γ-TLOgenes examined (i.e.,TLOγ5 and TLOγ11) had a far lower impact on complementing phenotypic and transcriptomic changes. Uniquely, expression ofTLOβ2in thetloΔmutant stimulated filamentous growth in YEPD medium and this phenotype was enhanced when Tloβ2 expression was increased to levels far in excess of Med3. In contrast, expression of reintegratedTLOgenes in atloΔ/med3Δdouble mutant background failed to restore any of the phenotypes tested, suggesting that complementation of these Tlo-regulated processes requires a functional Mediator tail module. Together, these data confirm the importance of Med2/Tlo in a wide range ofC.albicanscellular activities and demonstrate functional diversity within the gene family which may contribute to the success of this yeast as a coloniser and pathogen of humans.