Environment-induced same-sex mating in the yeast Candida albicans through the Hsf1-Hsp90 pathway

Abstract

While sexual reproduction is pervasive in eukaryotic cells, the strategies employed by fungal species to achieve and complete sexual cycles is highly diverse and complex. Many fungi, including Saccharomyces cerevisiae and Schizosaccharomyces pombe, are homothallic (able to mate with their own mitotic descendants) because of homothallic switching (HO) endonuclease-mediated mating-type switching. Under laboratory conditions, the human fungal pathogen Candida albicans can undergo both heterothallic and homothallic (opposite- and same-sex) mating. However, both mating modes require the presence of cells with two opposite mating types (MTLa/a and α/α) in close proximity. Given the predominant clonal feature of this yeast in the human host, both opposite- and same-sex mating would be rare in nature. In this study, we report that glucose starvation and oxidative stress, common environmental stresses encountered by the pathogen, induce the development of mating projections and efficiently permit same-sex mating in C. albicans with an "a" mating type (MTLa/a). This induction bypasses the requirement for the presence of cells with an opposite mating type and allows efficient sexual mating between cells derived from a single progenitor. Glucose starvation causes an increase in intracellular oxidative species, overwhelming the Heat Shock transcription Factor 1 (Hsf1)- and Heat shock protein (Hsp)90-mediated stress-response pathway. We further demonstrate that Candida TransActivating protein 4 (Cta4) and Cell Wall Transcription factor 1 (Cwt1), downstream effectors of the Hsf1-Hsp90 pathway, regulate same-sex mating in C. albicans through the transcriptional control of the master regulator of a-type mating, MTLa2, and the pheromone precursor-encoding gene Mating α factor precursor (MFα). Our results suggest that mating could occur much more frequently in nature than was originally appreciated and that same-sex mating could be an important mode of sexual reproduction in C. albicans.

Fig 1. Glucose starvation promotes efficient polarized cell growth and induces the expression of mating-related genes in MTLa/a cells of C. albicans.

(A) Morphologies of the laboratory strain GH1013 (MTLa/a) grown on YPD-K and YP-K media. 1 × 10⁵ cells were spotted on YPD-K and YP-K media and cultured at 25°C for five days. Scale bar for colonies (left panels), 2 mm; scale bar for cells (right panels), 10 μm. (B) Morphologies of three clinical C. albicans strains (MTLa/a) grown on YP-K medium at 30°C for four days. Scale bar for colonies (inset), 2 mm; scale bar for cells, 10 μm. (C) Relative expression levels of mating-related genes normalized to ACT1. Cells of GH1013 were used, and culture conditions were same as described in panel (A). Error bars represent standard errors of technical duplicates. *p < 0.05, two-tailed Student t test. Experiment was repeated in a biological replicate, and a representative image is shown. The numerical data are presented in S3 Data. ACT1, ACTin 1; Bar1, BARrier 1; FIG1, Factor-Induced Gene 1; FUS1, cell FUSion 1; MFA1, Mating type A1; MFα, Mating α factor precursor; MTL, Mating type locus; p, mating projection; STE2, STErile 2; YPD-K, yeast extract-peptone-glucose-K₂HPO₄; YP-K, yeast extract-peptone-K₂HPO₄.

Fig 2. Glucose starvation induces same-sex mating in C. albicans.

(A) Same-sex mating between two “a” strains (GH1013 and GH1350a). 1 × 10⁷ cells of each strain were mixed and cultured on YPD-K and YP-K media at 25°C. After three days of growth, the mating mixture was replated onto SCD-Arg, SCD-His, and SCD-Arg-His dropout plates to assess mating efficiency. The middle panel (up) indicates that two “a” cells underwent cell fusion and a daughter cell grew out from the conjunction tube. Scale bar, 10 μm. (B) PCR verification of the MTL types. Strains used: lane 1, SN152 (a/α); 2, GH1350 (α/α); 3, GH1350a (a/a); 4, GH1013 (a/a); 5–7, progeny strains of the GH1350a × GH1013a cross. (C) FACS analysis of the DNA content of parental and progeny strains. Parental diploids have the standard G1 and G2 cell cycle peaks representing 2C and 4C DNA levels. Mating progeny contain DNA content corresponding to 4C and 8C peaks, confirming their tetraploid nature. Arg, arginine; d, daughter cell; FACS, fluorescence-activated cell sorting; His, histidine; M, DNA ladder; MTL, Mating type locus; p, mating projection; SCD, synthetic complete medium; YPD-K, yeast extract-peptone-glucose-K₂HPO₄; YP-K, yeast extract-peptone-K₂HPO₄.

Fig 3. Oxidative stress promotes the development of mating projections in C. albicans.

(A) Relative ROS levels. 1 × 10⁵ cells of strain GH1013 were spotted on YPD-K and YP-K media and cultured at 25°C for one, two, three, or five days. For each day, 1 × 10⁶ cells were used for ROS level determination. Error bars represent standard deviation of three biological replicates. * indicates significant difference (p < 0.05, two-tailed Student t test). (B) H₂O₂ induces the development of mating projections in the presence of glucose. 200 μL of H₂O or 5 mM H₂O₂ solution was spread on YPD-K medium plates (90 mm). 1 × 10⁶ cells of strain GH1350a were spotted on the medium and cultured at 25°C for three or five days. Percentages of projected cells are indicated in the corresponding images. (C) Relative expression levels of mating-related genes in response to H₂O₂ treatments on YPD-K medium. Error bars, standard errors of technical duplicates. *p < 0.05, two-tailed Student t test. Experiment was performed in biological replicate, and a representative image is shown. (D) Efficiency of same-sex mating on YPD-K medium with or without H₂O₂ treatment. The mating mixtures were grown on different media at 25°C for seven days. To make YPD-K + H₂O₂ plates, 200 μL H₂O₂ (5 mM) was spread on the medium surface (of a 90-mm plate). The numerical data are presented in S3 Data. FIG1, Factor-Induced Gene 1; FUS1, cell FUSion 1; MFA1, Mating type A1; MFα, Mating α factor precursor; p, mating projection; ROS, reactive oxidative species; YPD-K, yeast extract-peptone-glucose-K₂HPO₄; YP-K, yeast extract-peptone-K₂HPO₄.

Fig 4. Global gene-expression–profile analysis of C. albicans in the presence and absence of glucose.

C. albicans cells were spotted and grown on YPD-K or YP-K media at 25°C for 60 hours. Total RNA was extracted and used for RNA-Seq assays. To be considered differentially expressed, a gene must satisfy three criteria: (1) an FPKM value higher than or equal to 20 at least in one sample, (2) a fold change value higher than or equal to 1.5, and (3) an adjusted p-value (FDR) lower than 0.05. (A) Venn diagram depicting relationships between differentially expressed genes on YPD-K (412) and YP-K (408) media and HSP90 genetic interactors (indicated in the ellipse). (B) Selected heat-shock-protein–encoding, oxidative-stress–induced, and mating-related genes up-regulated in YP-K medium. AIF1, Apoptosis-Inducing Factor 1; BAR1, BARrier 1; CEK1, Candida ERK-family protein kinase; ERK, ERK-family protein kinase; FDR, false discovery rate; FPKM, fragments per kb per million reads; GPI, glycosylphosphatidylinisotol; GST3, Glutathione S-transferase; HMX1, HeMe oXygenase; Hsp90, Heat shock protein 90; KAR2, KARyogamy; MAP, mitogen-activated protein; MFA1, Mating type A1; NADH, Nicotinamide adenine dinucleotide; orf19.3475, Candida gene orf19.3475; PST2, Protoplasts-SecreTed 1; RNA-Seq, RNA sequencing; SIS1, Slt4 Suppressor; SOD5, SuperOxide Dismutase 5; SSA2, Stress-Seventy subfamily A; STE4, STErile 4; YPD-K, yeast extract-peptone-glucose-K₂HPO₄; YP-K, yeast extract-peptone-K₂HPO₄.

Fig 5. Role of Hsf1 in the induction of mating projections in C. albicans.

(A) Morphologies of the control and tetON-HSF1/hsf1 mutant. 1 × 10⁵ cells were spotted on YPD-K and YP-K media without or with 40 μg/mL Dox and cultured at 25°C for three or five days. Percentages of projected cells are indicated in the corresponding images. Scale bar for colonies, 2 mm (inset); scale bar for cells, 10 μm. Control, GH1013cartTA. (B) Relative expression levels of mating-related genes. 1 × 10⁵ cells of each strain were cultured on YPD-K medium (for six days) or on YP-K medium (for three days) without or with 40 μg/mL Dox at 25°C. Error bars represent standard errors of technical duplicates. *p < 0.05, two-tailed Student t test. Experiment was repeated in a biological replicate, and a representative image is shown. The numerical data are presented in S3 Data. Dox, doxycycline; FIG1, Factor-Induced Gene 1; FUS1, cell FUSion 1; Hsf1, Heat Shock transcription Factor 1; MFA1, Mating type A1; MFα, Mating α factor precursor; p, mating projection; tetON, tetracycline-induced; tetON-HSF1/hsf1, tetON-promoter–controlled conditional expression strain of HSF1; WT, wild type; YPD-K, yeast extract-peptone-glucose-K₂HPO₄; YP-K, yeast extract-peptone-K₂HPO₄.

Fig 6. Down-regulation of Hsp90 promotes the development of mating projections.

(A) Morphologies of the control and tetON-HSP90/hsp90 mutant. Control, GH1013 + pACT1-WOR1; tetON-HSP90/hsp90, a tetON-promoter–controlled conditional expression strain of HSP90 with ectopically expressed WOR1 (+ pACT1-WOR1). 1 × 10⁵ cells were spotted on YPD-K and YP-K media with or without Dox as indicated and cultured at 25°C for three, five, or seven days. Percentages of projected cells are indicated in the corresponding images. Scale bar for colonies, 2 mm (inset); scale bar for cells, 10 μm. (B) Relative expression of mating-related genes. 1 × 10⁵ cells of each strain were cultured on YPD-K medium (for seven days) or on YP-K medium (for three days) with 40 μg/mL Dox at 25°C. Relative expression levels were not tested on YP-K medium without Dox since cell viability of the tetON-HSP90/hsp90 mutant was severely impaired. Error bars represent standard errors of technical duplicates. *p < 0.05, two-tailed Student t test. Experiment was repeated in a biological replicate, and a representative image is shown. The numerical data are presented in S3 Data. Dox, doxycycline; FIG1, Factor-Induced Gene 1; FUS1, cell FUSion 1; Hsp90, Heat shock protein 90; MFA1, Mating type A1; MFα, Mating α factor precursor; NA, not analyzed; p, mating projection; pACT1, plasmid pACT1; tetON, tetracycline-induced; WOR1, White–Opaque Regulator 1; YPD-K, yeast extract-peptone-glucose-K₂HPO₄; YP-K, yeast extract-peptone-K₂HPO₄.

Fig 7. Role of the Cwt1 transcription factor in the induction of mating projections.

(A and B) Morphologies of the control (GH1013 + ARG4 + HIS1) and cwt1/cwt1 mutant on YP-K (A) or YPD-K (B) medium. 1 × 10⁵ cells of each strain were spotted on YPD-K and YP-K media and cultured at 25°C for three or five days. Percentages of projected cells are indicated in the corresponding images. Scale bar for colonies, 2 mm (inset); scale bar for cells, 10 μm. (C) Relative expression levels of CWT1 in YPD-K and YP-K media. Cells of C. albicans were spotted on YPD-K and YP-K media and cultured at 25°C for five days. (D) Relative expression levels of CWT1 in the control (GH1013cartTA) and tetON-HSF1/hsf1 on YP-K medium. Error bars, standard errors of technical duplicates. *p < 0.05, two-tailed Student t test. Percentages indicate the ratio of gene expression in tetON-HSF1/hsf1 mutant relative to gene expression in control. The numerical data are presented in S3 Data. (E) Physical interaction of Cwt1 and Hsp90. Co-IP assays were performed using a strain with 13× Myc-tagged Cwt1 and GFP-tagged Hsp90. Lanes 1–4, samples co-immunoprecipitated by the GFP antibody Sepharose and analyzed by immunoblotting with the anti-Myc antibody. Lanes 5–8, whole-cell extracts analyzed by immunoblotting with the anti-Myc antibody. Experiment was performed in biological replicate, and a representative image is shown. Arg, arginine; Cwt1, Cell Wall Transcription factor 1; Dox, doxycycline; GFP, green fluorescent protein; His, histidine; Hsf1, Heat Shock transcription Factor 1; Hsp90, Heat shock protein 90; IP, immunoprecipitation; Myc, Myc epitope tag; p, mating projection; tetON, tetracycline-induced; tetON-HSF1/hsf1, tetON-promoter–controlled conditional expression strain of HSF1; YPD-K, yeast extract-peptone-glucose-K₂HPO₄; YP-K, yeast extract-peptone-K₂HPO₄.

Fig 8. MTLa2 regulates the development of mating projections in C. albicans.

(A) Relative expression levels of MTLa2 in the corresponding controls, tetON-HSF1/hsf1, tetON-HSP90/hsp90, and cwt1/cwt1 mutants, and MTLa2-overexpressing strain on YPD-K medium. Transcript levels were normalized to ACT1. Error bars, standard errors of technical duplicates. *p < 0.05, two-tailed Student t test. Experiment was performed in biological replicate, and a representative image is shown. (B) 1 × 10⁵ cells of each strain were spotted on YP-K medium and cultured at 25°C for five days. Morphologies of the control (WT + pACTS) and MTLa2-overexpressing strains (WT + pACTS-MTLa2) on YP-K medium. WT, GH1350a. Scale bar for colonies, 2 mm (inset); scale bar for cells, 10 μm. (C) Cwt1 binds to the promoters of MTLa2 and MFα. ChIP assays were performed in TAP-tagged Cwt1 strains. Percentages of input genomic DNA are indicated. 1 × 10⁵ cells of each strain were spotted on YP-K or YPD-K media and cultured at 25°C for one or three days. Dark arrows indicate detected promoter regions. d1, d2, and d3, three detected sites of MFα. Error bars represent standard error of two technical replicates. *p < 0.05, two-tailed Student t test. Experiment was performed in biological replicate with a representative image shown. The data for the untagged control correspond to samples harvested at day 1. The numerical data are presented in S3 Data. (D) Regulatory model of glucose-starvation–or oxidative-stress–induced same-sex mating in C. albicans. Glucose starvation or oxidative stresses cause the overwhelming of the Hsf1/Hsp90 functional capacity that regulates the transcriptional expression and activity of CWT1 in both direct and indirect manners. Cwt1 regulates same-sex mating through the control of MFα or MTLa2. ACT1, ACTin 1; ChIP, chromatin immunoprecipitation; Cta4, Candida TransActivating protein 4; Cwt1, Cell Wall Transcription factor 1; Dox, doxycycline; Hsf1, Heat Shock transcription Factor 1; Hsp90, Heat shock protein 90; MFα, Mating α factor precursor; MTL, Mating type locus; pACTS, plasmid pACTS; TAP, Tandem affinity purification; tetON, tetracycline-induced; tetON-HSF1/hsf1, tetON-promoter–controlled conditional expression strain of HSF1; WT, wild type; YPD-K, yeast extract-peptone-glucose-K₂HPO₄; YP-K, yeast extract-peptone-K₂HPO₄.

Fig 9

Potential autocrine (A) and paracrine (B) pheromone response models for stress-induced mating-projection formation and same-sex mating. Neither α-pheromone nor a-pheromone is constitutively expressed in “a” cells of C. albicans. Glucose starvation or oxidative stress first induces α-pheromone and its receptor Ste2 expression in “a” cells. α-pheromone binds to Ste2 and activates the pheromone-response pathway, which subsequently induces the expression of a-pheromone. “a” cells then become mating competent as both “a” and “α” types. The activation of the pheromone-response pathway promotes mating projection formation and same-sex mating. Autocrine pheromone response, self-activation (A); paracrine pheromone response, activation of neighbor cells (B). MFA1, Mating type A1; STE2, STErile 2.