A forkhead transcription factor is important for true hyphal as well as yeast morphogenesis in Candida albicans

Abstract

Candida albicans is an important pathogen of immunocompromised patients which grows with true hyphal, pseudohyphal, and yeast morphologies. The dynamics of cell cycle progression are markedly different in true hyphal relative to pseudohyphal and yeast cells, including nuclear movement and septin ring positioning. In Saccharomyces cerevisiae, two forkhead transcription factors (ScFKH1 and ScFKH2) regulate the expression of B-cyclin genes. In both S. cerevisiae and Schizosaccharomyces pombe, forkhead transcription factors also influence morphogenesis. To explore the molecular mechanisms that connect C. albicans morphogenesis with cell cycle progression, we analyzed CaFKH2, the single homolog of S. cerevisiae FKH1/FKH2. C. albicans cells lacking CaFkh2p formed constitutive pseudohyphae under all yeast and hyphal growth conditions tested. Under hyphal growth conditions levels of hyphae-specific mRNAs were reduced, and under yeast growth conditions levels of several genes encoding proteins likely to be important for cell wall separation were reduced. Together these results imply that Fkh2p is required for the morphogenesis of true hyphal as well as yeast cells. Efglp and Cphlp, two transcription factors that contribute to C. albicans hyphal growth, were not required for the pseudohyphal morphology of fkh2 mutants, implying that Fkh2p acts in pathways downstream of and/or parallel to Efglp and Cphlp. In addition, cells lacking Fkh2p were unable to damage human epithelial or endothelial cells in vitro, suggesting that Fkh2p contributes to C. albicans virulence.

FIG. 1.

Protein sequence comparison of C. albicans Fkh2p (CaFkh2p) and S. cerevisiae Fkh1 and Fkh2p (ScFkh1p, ScFkh2p). Percent identity/percent similarity values are shown for regions between dashed lines. Values were obtained by using the GAP program from the Wisconsin Package, version 10.2 (Genetics Computer Group, Madison, Wis.). Forkhead DNA-binding domains (forkhead) and forkhead-associated domains (FHA) are shown in black boxes. Asterisks represent positions of CUG codons in the CaFkh2p sequence. aa, amino acids.

FIG. 2.

CaFKH2 suppresses the cell morphology defect of an S. cerevisiae fkh1Δfkh2Δ strain. Strain CFY147 (fkh1Δfkh2Δ) was transformed with pYES2 (vector) or pYES-CaFKH2 (P_GAL1-CaFKH2) and grown in the presence of 2% glucose or 2% raffinose liquid media. Exponentially growing cells were viewed with DIC optics.

FIG. 3.

fkh2Δ/fkh2Δ cells form irregular colonies and pseudohyphal cells on yeast medium. (A) Colony morphology of fkh2Δ/fkh2Δ cells. YJB6284 (FKH2/FKH2), YJB6290 (fkh2Δ/FKH2), and YJB6292 (fkh2Δ/fkh2Δ) cells were grown on YPAD agar medium for 2 days at 30°C. (B and C) Cellular morphology of fkh2Δ/fkh2Δ cells. Strains depicted in panel A were grown in YPAD liquid medium to mid-logarithmic phase and were stained with Calcofluor White to visualize chitin distribution. DIC images (B) and fluorescence micrographs (C) are shown.

FIG. 4.

P_MET3-FKH2 restores wild-type cellular morphology to fkh2 mutant cells. YJB6284 (FKH2/FKH2), YJB6292 (fkh2Δ/fkh2Δ), YJB6403 (fkh2Δ/fkh2ΔRP10::P_MET3-FKH2), and YJB4828 (fkh2Δ/P_MET3-FKH2) cells were grown to saturation in SDC-methionine-cysteine-uridine at 30°C. Cells were diluted into either SDC-methionine-cysteine-uridine (−Met/Cys) or SDC plus 10 mM methionine plus 2 mM cysteine-uridine (+Met/Cys) and were grown overnight at 30°C. DIC images are shown.

FIG. 5.

fkh2Δ/fkh2Δ cells are defective in cell separation. Transmission electron micrograph of YJB6292 (fkh2Δ/fkh2Δ) cells grown in YPAD liquid medium to mid-logarithmic phase at 30°C. White arrows indicate plasma membrane (PM).

FIG. 6.

Fkh2p affects the formation of true hyphae. YJB6284 (FKH2/FKH2), YJB6290 (fkh2Δ/FKH2), and YJB6292 (fkh2Δ/fkh2Δ) cells were grown under different hyphae-inducing conditions. For the panels labeled serum and GlcNAc, cells were grown to saturation in YPAD at 30°C, diluted into YPAD plus 10% serum or YPA plus 4 mM GlcNAc, and incubated for 4 h. For the panel labeled M199, cells were grown to saturation in M199 (pH 4.0 at 37°C), diluted into M199 (pH 8.0), and incubated at 37°C for 4 h. For the panel labeled Spider, cells were grown to saturation in YPAD at 30°C, plated onto solid Spider medium, and incubated for 7 days at 30°C. For the panel labeled milk-Tween, cells were grown to saturation in YPAD at 30°C, plated onto milk-Tween agar plates, and incubated for 7 days at 37°C. For the panel labeled embedded, cells were grown to saturation in YPAD at 30°C, diluted into cooled, molten YPS agar, and incubated for 3 days at 28°C. Arrows indicate septal constrictions.

FIG. 7.

Epistasis analysis of fkh2. CAF2 (FKH2/FKH2 CPH1/CPH1 EFG1/EFG1), JKC19 (cph1Δ/cph1Δ), HLC52 (efg1Δ/efg1Δ), HLC54 (cph1Δ/cph1Δefg1Δ/efg1Δ), YJB6292 (fkh2Δ/fkh2Δ), YJB6441 (fkh2Δ/fkh2Δcph1Δ/cph1Δ), YJB6413 (fkh2Δ/fkh2Δefg1Δ/efg1Δ), and YJB5802 (fkh2Δ/fkh2Δefg1Δ/efg1Δcph1Δ/cph1) cells were grown to mid-logarithmic phase at 30°C in YPAD. DIC micrographs are shown.

FIG. 8.

Fkh2p-GFP localizes to the nucleus. A strain expressing GFP-tagged Fkh2p (YJB5535) was grown overnight in SDC containing 1 μg of DAPI/ml. Cells were diluted into SDC containing 1 μg of DAPI/ml and were grown at 30°C to mid-logarithmic phase. DIC and fluorescent micrographs were taken of unfixed cells. DAPI images were false-colored red, and Fkh2p-GFP images were false-colored green for merging.

FIG. 9.

Fkh2p affects transcription of a subset of genes. (A to C) A total of 319 ORFs on a C. albicans cDNA microarray were competitively hybridized with Cy3- and Cy5-labeled cDNA derived from the following strains: (1) YJB6284 (FKH2/FKH2) grown for 3 h in YPAD plus 10% serum at 37°C and YJB6284 grown in YPAD at 30°C; (2) YJB6292 (fkh2Δ/fkh2Δ) grown for 3 h in YPAD plus 10% serum at 37°C and YJB6284 grown for 3 h in YPAD plus 10% serum at 37°C; and (3) YJB6292 grown in YPAD at 30°C and YJB6284 grown in YPAD at 30°C. Fold induction or repression is indicated by the color scale where red indicates an increase and green indicates a decrease in expression. Black indicates a less than twofold change. (A) Genes with increased expression in FKH2/FKH2 but not fkh2Δ/fkh2Δ cells when grown under hyphae-inducing conditions. (B) Genes with decreased expression in fkh2Δ/fkh2Δ cells compared to that of FKH2/FKH2 cells grown under yeast growth conditions. (C) Genes with increased expression in fkh2Δ/fkh2Δ cells compared to that of FKH2/FKH2 cells grown under yeast growth conditions. Asterisks indicate C. albicans genes similar to S. cerevisiae genes that display altered expression in S. cerevisiae fkh1 fkh2 mutants. (D) Northern analysis is consistent with microarray data. Northern analysis of RNA from YJB6284 and YJB6292 grown in the absence (−) or presence (+) of serum, as described above, is depicted. CYB4, ECE1, and HWP1 probes were prepared as described in Materials and Methods. rRNA was used as a loading control.

FIG. 10.

Fkh2p is required for epithelial and endothelial cell injury. The HOK-16B-BaP-T1 oral epithelial cell line and human umbilical vein endothelial cells were incubated with YJB6284 (FKH2/FKH2), YJB6288 (fkh2Δ/FKH2), and YJB6292 (fkh2Δ/fkh2Δ) cells. The extent of epithelial cell damage was measured with a chromium release assay. Results are the averages ± standard deviations of triplicate measurements. Asterisks indicate a P value of <0.001 compared to that for cells infected with the FKH2/FKH2 strain by analysis of variance.