Deletion of the Candida albicans TLO gene family using CRISPR-Cas9 mutagenesis allows characterisation of functional differences in -, - and - TLO gene function

The Candida albicans genome contains between ten and fifteen distinct TLO genes that all encode a Med2 subunit of Mediator. In order to investigate the biological role of Med2/Tlo in C. albicans we deleted all fourteen TLO genes using CRISPR-Cas9 mutagenesis. ChIP-seq analysis showed that RNAP II localized to 55% fewer genes in the tlo Delta mutant strain compared to the parent, while RNA-seq analysis showed that the tlo Delta mutant exhibited differential expression of genes required for carbohydrate metabolism, stress responses, white opaque switching and filamentous growth. Consequently, the tlo Delta 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 alpha-, beta-and gamma-TLO clades resulted in the complementation of the mutant phenotypes, but to different degrees. TLO alpha 1 could 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 gamma-TLO genes examined (i.e., TLO gamma 5 and TLO gamma 11) had a far lower impact on complementing phenotypic and transcriptomic changes. Uniquely, expression of TLO beta 2 in the tlo Delta mutant stimulated filamentous growth in YEPD medium and this phenotype was enhanced when Tlo beta 2 expression was increased to levels far in excess of Med3. In contrast, expression of reintegrated TLO genes in a tlo Delta/ med3 Delta double 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 of C. albicans cellular 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.