A CUG codon-adapted anchor-away toolkit for functional analysis of genes in Candida albicans

Promoter shutoff of essential genes in the diploid Candida albicans has often been insufficient to create tight, conditional null alleles due to leaky expression and has been a stumbling block in pathogenesis research. Moreover, homozygous deletion of non-essential genes has often been problematic due to the frequent aneuploidy in the mutant strains. Rapid, conditional depletion of essential genes by the anchor-away strategy has been successfully employed in Saccharomyces cerevisiae and other model organisms. Here, rapamycin mediates the dimerization of human FK506-binding protein (FKBP12) and FKBP12-rapamycin-binding (FRB) domain-containing target protein, resulting in relocalization to altered sub-cellular locations. In this work, we used the ribosomal protein Rpl13 as the anchor and took two nuclear proteins as targets to construct a set of mutants in a proof-of-principle approach. We first constructed a rapamycin-resistant C. albicans strain by introducing a dominant mutation in the CaTOR1 gene and a homozygous deletion of RBP1, the ortholog of FKBP12, a primary target of rapamycin. The FKBP12 and the FRB coding sequences were then CUG codon-adapted for C. albicans by site-directed mutagenesis. Anchor-away strains expressing the essential TBP1 gene or the non-essential SPT8 gene as FRB fusions were constructed. We found that rapamycin caused rapid cessation of growth of the TBP-AA strain within 15 minutes and the SPT8-AA strain phenocopied the constitutive filamentous phenotype of the spt8Δ/spt8Δ mutant. Thus, the anchor-away toolbox for C. albicans developed here can be employed for genome-wide analysis to identify gene function in a rapid and reliable manner, further accelerating anti-fungal drug development in C. albicans.

Molecular genetic studies thus far have identified ~27% open-reading frames as being essential for the vegetative growth of Candida albicans in rich medium out of a total 6,198 haploid set of open reading frames. However, a major limitation has been to construct rapid conditional alleles of essential C. albicans genes with near quantitative depletion of encoded proteins. Here, we have developed a toolbox for rapid and conditional depletion of genes that would aid studies of gene function of both essential and non-essential genes.