Role of transcription factor CaNdt80p in cell separation, hyphal growth, and virulence in Candida albicans

Abstract

The NDT80/PhoG transcription factor family includes ScNdt80p, a key modulator of the progression of meiotic division in Saccharomyces cerevisiae. In Candida albicans, a member of this family, CaNdt80p, modulates azole sensitivity by controlling the expression of ergosterol biosynthesis genes. We previously demonstrated that CaNdt80p promoter targets, in addition to ERG genes, were significantly enriched in genes related to hyphal growth. Here, we report that CaNdt80p is indeed required for hyphal growth in response to different filament-inducing cues and for the proper expression of genes characterizing the filamentous transcriptional program. These include noteworthy genes encoding cell wall components, such as HWP1, ECE1, RBT4, and ALS3. We also show that CaNdt80p is essential for the completion of cell separation through the direct transcriptional regulation of genes encoding the chitinase Cht3p and the cell wall glucosidase Sun41p. Consistent with their hyphal defect, ndt80 mutants are avirulent in a mouse model of systemic candidiasis. Interestingly, based on functional-domain organization, CaNdt80p seems to be a unique regulator characterizing fungi from the CTG clade within the subphylum Saccharomycotina. Therefore, this study revealed a new role of the novel member of the fungal NDT80 transcription factor family as a regulator of cell separation, hyphal growth, and virulence.

Fig. 1.

The NDT80/PhoG transcription factor family across Ascomycota.ic> (A) Functional-domain organizations of NDT80/PhoG family members in Saccharomyces cerevisiae and Candida albicans. Both the DNA-binding domain (Ndt80p DBD) and putative glutamine-rich activation domain (Ndt80p AD) are represented. (B) Structural organizations of NDT80/PhoG family members across different classes of ascomycetes. The topology of the tree was based on Fitzpatrick et al. (12). The CTG clade is highlighted by a red box. WGD identifies the clade containing species in which whole-genome duplication has occurred.

Fig. 2.

Ndt80p is required for cell separation completion. (A) Images of wt (DAY286), ndt80/ndt80 mutant (AS31), and revertant ndt80/ndt80/NDT80 (AS33) strains showing the cell separation defect. The cells were stained with calcofluor white. (B) Quantification of the cell separation defect. Shown are the percentages of chains of cells containing 1 or 2, 3 or 4, 5 or 6, or more than 6 cells.

Fig. 3.

Ndt80p directly regulates genes implicated in cell separation. (A) Relationship between Ndt80p-bound genes and genes showing altered expression in the ndt80 mutant (AS31). (B) Ndt80p promoter occupancies of the endochitinase Cht3p and the glucosidase Sun41p as determined by Sellam et al. (36). The positions of the middle sporulation element motifs are shown.

Fig. 4.

The ndt80 cell separation defect is reverted by the overexpression of CHT3 or SUN41. (A) Average transcript levels of CHT3 and SUN41 in overexpression mutants relative to the wt strain (DAY286). For each gene, two clones (clones C1 and C20 for the ndt80/Act::SUN41 strain and clones C1 and C11 for the ndt80/Act::CHT3 strain) were evaluated. The reported values are the means of two independent experiments. (B) Evaluation of the percentages of chains of cells with 1 or 2, 3 or 4, 5 or 6, or more than 6 cells in overexpressing strains.

Fig. 5.

CaNdt80p is essential for hyphal growth in response to different filament-inducing conditions. (A and B) Cell morphologies of wt (DAY286), ndt80/ndt80 mutant (AS31), and revertant ndt80/ndt80/NDT80 (AS33) strains on liquid media supplemented with fetal bovine serum (A) or N-acetyl-d-glucosamine (B). (C and D) Colony morphologies of wt, ndt80, and revertant strains after 5 days of growth on solid media under hypoxic conditions (C) or in M199 medium at pH 8 (D).

Fig. 6.

CaNdt80p is required for the activation of hypha-specific genes. Shown are the overlaps between genes differentially regulated during the yeast-to-hypha switch as determined by Nantel et al. (29) and Kadosh et al. (17) and genes downregulated (A) or upregulated (B) in a wt-versus-ndt80 comparison.

Fig. 7.

Ndt80p is required for full virulence in a B6 mouse model. (A) Survival of mice infected with ndt80 mutant, ndt80 revertant, and wt parental strains. Mice were inoculated by tail vein injection, and survival was measured over a 21-day period. (B) The kidney fungal load was determined 7 days after injection. The error bars indicate standard deviations.