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. IMPORTANCE: 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.

Reconfiguration of Transcriptional Control of Lysine Biosynthesis in Candida albicans Involves a Central Role for the Gcn4 Transcriptional Activator.

Evolution of transcriptional control is essential for organisms to cope with diversification into a spectrum of environments, including environments with limited nutrients. Lysine biosynthesis in fungi occurs in eight enzymatic steps. In Saccharomyces cerevisiae, amino acid starvation elicits the induction of LYS gene expression, mediated by the master regulator Gcn4 and the pathway-specific transcriptional regulator Lys14. Here, we have shown that the activation of LYS gene expression in the human fungal pathogen Candida albicans is predominantly controlled by Gcn4 under amino acid starvation conditions. Multiple lines of study showed that the four C. albicans LYS14-like genes have no role in the regulation of lysine biosynthesis. Whereas Gcn4 is dispensable for the growth of S. cerevisiae under lysine deprivation conditions, it is an essential regulator required for the growth of C. albicans under these conditions, as gcn4 deletion caused lysine auxotrophy. Gcn4 is required for the induction of increased LYS2 and LYS9 mRNA but not for the induction of increased LYS4 mRNA. Under lysine or isoleucine-valine deprivation conditions, Gcn4 recruitment to LYS2 and LYS9 promoters was induced in C. albicans. Indeed, in contrast to the S. cerevisiae LYS gene promoters, all LYS gene promoters in C. albicans harbored a Gcn4 binding site but not all harbored the S. cerevisiae Lys14 binding site, indicating the evolutionary divergence of cis-regulatory motifs. Thus, the transcriptional rewiring of the lysine biosynthetic pathway in C. albicans involves not only neofunctionalization of the four LYS14-like genes but the attendant strengthening of control by Gcn4, indicating a coordinated response with a much broader scope for control of amino acid biosynthesis in this human pathogen. IMPORTANCE Microbes evolve rapidly so as to reconfigure their gene expression to adapt to the metabolic demands in diverse environmental niches. Here, we explored how conditions of nutrient deprivation regulate lysine biosynthesis in the human fungal pathogen Candida albicans. We show that although both Saccharomyces cerevisiae and C. albicans respond to lysine deprivation by transcriptional upregulation of lysine biosynthesis, the regulatory factors required for this control have been reconfigured in these species. We found that Gcn4 is an essential and direct transcriptional regulator of the expression of lysine biosynthetic genes under lysine starvation conditions in C. albicans. Our results therefore suggest that the regulation of the lysine biosynthetic pathway in Candida clade genomes involves gain of function by the master transcriptional regulator Gcn4, coincident with the neofunctionalization of the S. cerevisiae pathway-specific regulator Lys14.