G1/S transcription factor orthologues Swi4p and Swi6p are important but not essential for cell proliferation and influence hyphal development in the fungal pathogen Candida albicans

Abstract

The G(1)/S transition is a critical control point for cell proliferation and involves essential transcription complexes termed SBF and MBF in Saccharomyces cerevisiae or MBF in Schizosaccharomyces pombe. In the fungal pathogen Candida albicans, G(1)/S regulation is not clear. To gain more insight into the G(1)/S circuitry, we characterized Swi6p, Swi4p and Mbp1p, the closest orthologues of SBF (Swi6p and Swi4p) and MBF (Swi6p and Mbp1p) components in S. cerevisiae. The mbp1Δ/Δ cells showed minor growth defects, whereas swi4Δ/Δ and swi6Δ/Δ yeast cells dramatically increased in size, suggesting a G(1) phase delay. Gene set enrichment analysis (GSEA) of transcription profiles revealed that genes associated with G(1)/S phase were significantly enriched in cells lacking Swi4p and Swi6p. These expression patterns suggested that Swi4p and Swi6p have repressing as well as activating activity. Intriguingly, swi4Δ/Δ swi6Δ/Δ and swi4Δ/Δ mbp1Δ/Δ strains were viable, in contrast to the situation in S. cerevisiae, and showed pleiotropic phenotypes that included multibudded yeast, pseudohyphae, and intriguingly, true hyphae. Consistently, GSEA identified strong enrichment of genes that are normally modulated during C. albicans-host cell interactions. Since Swi4p and Swi6p influence G(1) phase progression and SBF binding sites are lacking in the C. albicans genome, these factors may contribute to MBF activity. Overall, the data suggest that the putative G(1)/S regulatory machinery of C. albicans contains novel features and underscore the existence of a relationship between G(1) phase and morphogenetic switching, including hyphal development, in the pathogen.

Fig. 1.

Deletion of SWI4 or SWI6 results in dramatic changes in growth pattern, including cell enlargement and induction of fila-ments, in contrast to deletion of MBP1. (A) Cells from strains BH440 (MBP1/MBP1 SWI6/SWI6 SWI4/SWI4 URA3⁺ HIS1⁺), BH261 (mbp1Δ::URA3/mbp1Δ::HIS1), BH120 (swi6Δ::URA3/swi6Δ::HIS1), and BH185 (swi4Δ::URA3/swi4Δ::HIS1) were incubated in minimal medium overnight and then diluted into fresh medium and incubated for 7 h at 30°C. (B) Strains BH120, BH185, and BH440 at higher magnification. Bar = 10 μm.

Fig. 2.

Distribution of yeast size, represented by length-to-width measurements (μm²), in cells lacking Swi4p, Swi6p, or Mbp1p. Cells from strains BH440 (MBP1/MBP1 SWI6/SWI6 SWI4/SWI4 URA3⁺ HIS1⁺), BH261 (mbp1Δ::URA3/mbp1Δ::HIS1), BH120 (swi6Δ::URA3/swi6Δ::HIS1), and BH185 (swi4Δ::URA3/swi4Δ::HIS1) were incubated in liquid glucose minimal medium overnight and then diluted into fresh medium and incubated for 7 h at 30°C. Strain BH190 (swi6Δ::URA3/swi6Δ::HIS1 swi4Δ::hisG/MET3::SWI4-ARG4) was incubated in repressing medium for 7 h.

Fig. 3.

Depletion of both Swi4p and Swi6p or Swi4p and Mbp1p results in viable cells with phenotypes similar to those of swi4Δ/Δ or swi6Δ/Δ cells. (A) Cells from strains BH420 (SWI6/SWI6 SWI4/SWI4 URA3⁺ HIS1⁺ ARG4+), BH190 (swi6Δ::URA3/swi6Δ::HIS1 swi4Δ::hisG/MET3::SWI4-ARG4), and BH277 (mbp1Δ::URA3/mbp1Δ::HIS1 swi4Δ::hisG/MET::SWI4-ARG4) were incubated in inducing (−MC) or repressing medium (+MC) for 7 h at 30°. (B) Strains AG168 (swi4Δ::hisG/swi4Δ::URA3 swi6Δ::ARG4/swi6Δ::HIS1), HH62 (swi4Δ::hisG/swi4Δ::URA3 mbp1Δ::ARG4/mbp1Δ::HIS1), and BH420 were cultured in minimal medium overnight and then diluted into fresh medium and incubated for 7 h at 30°C.

Fig. 4.

Gene expression patterns in cells depleted of Swi6p and Swi4p. (A) Northern analysis demonstrating downregulation of G₁ cyclins CCN1 and PCL2 in cells depleted of Swi6p and Swi4p. A total of 20 μg of RNA from strains BH420 (SWI4/SWI4 SWI6/SWI6 URA3⁺ HIS1⁺ ARG4⁺), AG168 (swi4Δ::hisG/swi4Δ::URA3 swi6Δ::ARG4/swi6Δ::HIS1), and BH190 (swi6Δ::URA3/swi6Δ::HIS1 swi4Δ::hisG/MET3::SWI4-ARG4) grown under repressing conditions was loaded on the gel. ACT1 was used a loading control. (B) Venn diagrams comparing significantly modulated genes in strain BH190 with the total number of genes in C. albicans that show cell cycle-dependent periodic expression patterns (26). Strains BH190 and BH420 were incubated in repressing medium for 7 h and processed for microarray analysis. Data obtained from four microarray chips representing four separate samples were normalized with Lowess using GeneSpring software. Significantly modulated genes were determined based on a 1.7-fold cutoff and t test function with a P value of <0.05. (C) Pie categorization of significantly modulated genes in cells depleted of Swi4p and Swi6p. Gene names and functions were identified through GeneSpring analysis and updated using the Candida Genome Database (CGD) at http://candidagenome.org/. Different colors represent different categories as indicated. Genes were manually categorized according to a single function, although several have multiple functions.

Fig. 5.

Gene set enrichment analysis (GSEA) of the transcriptional profile of Swi4p- and Swi6p-depleted cells. A ranked list of genes modulated in Swi4p- and Swi6p-depleted cells was compared to 29 gene sets of 64 to 558 genes that exhibit cell cycle-dependent periodic expression in C. albicans opaque cells (26) or significant changes during polar morphogenesis, including yeast-to-hypha transitions and the production of highly elongated buds due to M or S phase arrest (5, 34, 43, 53, 58, 71). Genes in the ranked list are organized along the x axis with upregulated genes to the left and downregulated genes to the right. The positions of the genes in each gene set are illustrated by the vertical black bars, while the green curve represents the cumulative value of the enrichment score (y axis). Graphs of selected results are shown, while the complete GSEA output folder is included as GSEA data file S1 in the supplemental material. BMDM, bone marrow-derived monocytes; RHE90, reconstituted human epithelial cells; HU, hydroxyurea.

Fig. 6.

Yeast cells lacking Swi4p and/or Swi6p show pleiotropic changes in morphology and form true hyphae. (A) Cells of strain BH190 (swi6Δ::URA3/swi6Δ::HIS1 swi4Δ::hisG/MET3::SWI4-ARG4) (a, b), BH185 (swi4Δ::URA3/swi4Δ::HIS1) (d to g), and BH420 (SWI4/SWI4 SWI6/SWI6 URA3⁺ HIS1⁺ ARG4⁺) (c) were incubated for the indicated times in repressing medium at 30°C, fixed, and then stained with DAPI and calcofluor. Arrowheads indicate subapical, unconstricted septa, thin arrows show the first septa positioned away from the bud neck, and thick arrows demonstrate the first septa at the bud neck. (B) Strain BH420 was incubated for 3.0 h in the presence of 10% serum at 37°C for comparison of hyphal characteristics. Bars = 10 μm.