Media and reagents

Synthetic complete dextrose medium (SCD) and yeast extract peptone dextrose medium (YPD) were made as described previously [19]. YPD plates containing 200 µg/ml nourseothricin were used for selection of strains that were resistant to nourseothricin (Werner Bioagents, Jena, Germany) as previously described [20]. SCD low phosphate (LP) medium was made with yeast nutrient base (YNB) w/o phosphate (cat. #CYN6701, Formedium LTD, Hunstanton, England) and KH₂PO₄ was supplemented to a final concentration of 10 µM. LP medium was adjusted to pH 4.7 with 3 M HCl before autoclaving. Synthetic low ammonium dextrose (SLAD) medium was made as described previously [21]; 2% agar was washed five times with distilled water, autoclaved with 1.7 g/L YNB w/o ammonia, and then supplemented with ammonium sulfate and dextrose to final concentrations of 50 µM and 2%, respectively. Sorbitol medium (SOR) was SCD supplemented with 1 M sorbitol. Minimal (MIN) medium consisted of 7 g/L YNB and 2% dextrose. Spider medium contained 1.35% agar, 1% nutrient broth, 0.4% potassium phosphate, and 2% mannitol (pH 7.2). N-acetyl glucosamine containing medium (GlcNAc) was modified Lee's medium [22] without glucose but supplemented with 1.25% N-acetyl glucosamine (Sigma). Neutral pH medium was made by buffering SCD with 150 mM HEPES (pH 7.0). Calcofluor white stain was obtained from Fluka Biochemika and geldanamycin from A.G. Scientific, Inc.

Plasmids and strains

C. albicans strains used in this study are listed in Table 1 and oligonucleotides in Table S1. All strains are derived from SC5314 unless stated otherwise. To construct the pACT1-WOR1 plasmid, the promoter of the ACT1 gene was PCR amplified with oligonucleotides 610 and 611 and the WOR1 ORF with oligonucleotides 2421 and 2422. The two fragments were then combined in a single fusion PCR reaction using oligos 610 and 2422 and cloned into plasmid pSFS2A [20] between ApaI and XhoI restriction sites. A modified pSFS2A plasmid was also constructed in which the SAT1 gene was replaced with the gene for hygromycin B resistance. A region of plasmid pSFS2A was PCR amplified with oligos 1669 and 1652 and the gene for hygromycin B resistance amplified from plasmid pYM70 [23] with oligos 1651/1653. The two PCR products were combined by fusion PCR using oligos 1669/1651 and cloned between HindIII and PstI restriction sites in the pSFS2A backbone. The resulting pACT1-WOR1 plasmids (either with SAT1 or HYG markers) were digested with BglII to linearize in the pACT1 region and integrated into the endogenous ACT1 locus to obtain constitutive WOR1 expression. Correct integration was confirmed by PCR.

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Table 1. Strains used in this study.

https://doi.org/10.1371/journal.ppat.1003210.t001

To target RFG1 for deletion, a fusion PCR product was created as described previously [24]. Briefly, oligos 984/992 and 985/993 were used to PCR the 5′ and 3′ homologous flanks of RFG1 and these flanks combined with a selectable marker (LEU2) by fusion PCR [24]. The pSFS2 (rfg1::SAT1 flipper) plasmid [25] (a gift from David Kadosh, University of Texas San Antonio) was used to delete the second copy of RFG1 and generate rfg1::LEU2/rfg1::SAT1 double deletion mutants, as previously described [25]. The SAT1 marker was subsequently excised by growth on maltose medium [20] to obtain strain CAY2214. Fusion PCR was also used to delete CPH1 or EFG1 from CAY2214, using the selectable markers HIS1 and ARG4 to generate rfg1/cph1 and rfg1/efg1 double mutants. Oligos 982/990 and 983/991 were used to PCR amplify the 5′ and 3′ flanks of the CPH1 gene, and oligos 1215/2425 and 1216/1217 used to amplify the 5′ and 3′ flanks of the EFG1 gene. The MTLα locus was also deleted from efg1, czf1 and cph2 mutant strains acquired from the Fungal Genetics Stock Center (strains originally generated by Homann et al. [26]) using the plasmid pRB102, as previously described [27]. The SAT1 marker was subsequently excised by growth on maltose medium [20] to generate MTLa/MTLαΔ::FRT strains. Correct integration of constructs was verified by PCR across 5′ and 3′ disruption junctions, and loss of the ORF was confirmed with primers internal to the open reading frame. Auxotrophic strains were also transformed with C. albicans LEU2, HIS1, or ARG4, PCR amplified by oligos 2490/2491, 2492/2493 or 2494/2495, respectively, to restore prototrophy to these strains.

White- and opaque-specific reporter constructs were generated as follows. For the opaque-reporter, the SAT1 gene was PCR amplified from pSFS2A [20] using primers 169/170 and cloned into pCR-Blunt II-TOPO (Invitrogen) between XhoI and XbaI restriction sites. A triple mCherry reporter was next integrated into this vector by PCR amplifying three copies of the mCherry gene from plasmid pADH77 [28] with oligonucleotides 1846/1847, 1848/1849 and 1850/1386. The three mCherry PCR products were stitched together using BamHI/AflII, AflII/StuI, and StuI/SalI restriction sites and integrated between BamHI and XhoI sites in the vector backbone to generate plasmid pRB224. The C. albicans OP4 promoter was then PCR amplified using oligos 1974/1975 and cloned between KpnI and SacI restriction sites in pRB224 to generate the final reporter construct pRB227. This plasmid was linearized in the OP4 gene with BsgI and transformed into C. albicans as an opaque-specific reporter. For the white-cell reporter, the C. albicans WH11 gene promoter was PCR amplified with oligos 1384/1396 and the GFP gene amplified from pADH76 [28] with oligos 1385/1386. These PCR products were fused by PCR with oligos 1384 and 1386, digested with ApaI/SalI and cloned into pSFS2A to generate pRB168. This plasmid was linearized within the WH11 gene with AatII and transformed into C. albicans to generate a white-specific reporter.

An EFG1 complementation plasmid was constructed by PCR amplification of EFG1 using oligos 1838/1839, and cloning between ApaI and KpnI sites in the modified pSFS2A for hygromycin B resistance. The resulting EFG1 addback plasmid pRB326 was linearized by HpaI and integrated into the endogenous EFG1 locus in the efg1 mutant strain CAY3292. Similarly, oligos 1832/1833 were used to PCR amplify UME6 and the PCR product cloned between ApaI and KpnI sites in pSFS2A (hygromycin B) to generate plasmid pRB328. This plasmid was linearized by SmaI and integrated into the endogenous UME6 locus in the opaque ume6 mutant strain CAY1571 to obtain strain CAY3697.

Construction of a UME6 overexpressing strain was achieved using derivatives of strain MBY208, a gift of David Kadosh. MBY208 contains a construct expressing high levels of an Escherichia coli tet repressor-Saccharomyces cerevisiae Hap4 activation domain fusion protein, as well as a second construct expressing the UME6 gene under the control of the E. coli tet operator [29]. The MTLα locus was deleted in MBY208 using a derivative of pRB102 (contains hygromycin B marker) to generate white (CAY4504) and opaque (CAY4502) strains.

RNA sample preparation

Single opaque or white colonies were inoculated into liquid LP and SOR media at room temperature. Cells were harvested by centrifugation after 12 hours (LP) or 16 hours (SOR), and pellets frozen in liquid nitrogen. Total RNA was extracted from cell pellets (8–10 OD) following the RiboPure-Yeast Kit protocol (Applied Biosystems, Bedford, MA). RNA was treated with DNaseI (Applied Biosystems) to eliminate DNA contamination and re-extracted with phenol/chloroform. For quality control, RNA was analyzed using an Agilent 2100 Bioanalyzer to check RNA integrity.

Hybridization of cDNA to microarrays and data analysis

Aminoallyl-labeled cDNA synthesis and hybridization to microarrays was previously described by Tuch et al. [30]. Arrays were scanned on a GenePix 4000 scanner (Axon Instruments), data quantified using GENEPIX PRO version 3.0 and normalized using Goulphar (http://transcriptome.ens.fr/goulphar). Pairwise average linkage clustering analysis was performed using CLUSTER and visualized by TREEVIEW [31]. Significance Analysis of Microarrays (SAM, http://www-stat.stanford.edu/~tibs/SAM/) [32] and R (Ver 2.15.2, http://www.r-project.org/) were used to screen the statistically significant genes induced in filamentous opaque cells in LP or SOR medium versus SCD medium (four replicas each). The parameters used for screening and SAM results are provided in the supplemental data (see tables in Text S1 and Text S2). The Candida genome database (www.candidagenome.org) and the Yeast Genome Database (http://www.yeastgenome.org/) were used to facilitate further analysis. Array data has been uploaded to GEO (accession number GSE42963).

Sample preparation and light microscopy

White and opaque cells were streaked onto thin agar plates. After 22 hours of growth, a small square (∼1 cm²) was cut from the plate and stained with calcofluor white. Digital images of cells were collected with Infinity analyzer software and an Infinity 2 digital camera (Lumenera Corporation, Ottawa, Canada). DIC and fluorescent images were collected with a Zeiss Inverted Microscope (Axio Observer. Z1) fitted with an AxioCam HR. Images were processed with AxioVision Rel. 4.8 (Zeiss, Germany).

Scanning electron microscopy

C. albicans opaque cells were grown on solid SOR or LP media at 25°C for 22 hours. C. albicans white cells were grown in liquid YPD + 10% serum at 37°C for 2 hours. Cells were resuspended in water and attached to poly-L-lysine coated-coverslips. Cells were fixed with 2.5% (w/v) glutaraldehyde in 0.1 M Na-cacodylate buffer, pH 7.4 at 4°C, and washed with 0.1 M Na-cacodylate buffer, pH 7.4. The cells were postfixed with 1% aqueous osmium tetroxide in 0.1 M Na-cacodylate buffer, pH 7.4 at 25°C for 90 minutes, and washed with 0.1 M Na-cacodylate buffer, pH 7.4. Following fixation, cells were dehydrated gradually using a gradient ethanol series and subsequently dried in a critical point dryer. The samples were then coated with 20 nm gold palladium (60∶40) in an Emitech K550 sputter coater. Cells were imaged with a Hitachi S-2700 scanning electronic microscopy and collected with Quartz PCI software.

Opaque cells do not undergo filamentation under conditions that induce hyphal growth in white cells

Filamentation of C. albicans white cells occurs in response to a wide variety of environmental stimuli, including neutral pH, serum, nutrient limitation, and CO₂ [2], [5]. We first addressed whether C. albicans opaque cells can undergo filamentous growth under any of the established conditions that promote white cell filamentation. As opaque cells are unstable at 37°C in vitro [9], MTLa/a strains were locked in the opaque state by expressing the WOR1 gene under a constitutive promoter. WOR1 is the master regulator of the white-opaque switch, and constitutive expression of WOR1 ensures that cells are stably maintained in the opaque state [33]–[35]. As shown in Figure 1, white cells efficiently formed hyphae when grown in YPD medium supplemented with serum at 37°C, in low-nutrient Spider medium at 30° or 37°C, or in neutral pH Lee's medium at 37°C. Growth in YPD medium at 37°C was also sufficient to induce filamentous growth in white cells, as previously observed [36] (Figure 1B). In contrast, opaque cells did not undergo efficient filamentation under any of these culture conditions, and instead continued to grow predominantly as budding yeast cells (Figure 1). For example, > 99% of white cells grown in YPD+serum medium underwent filamentation, while less than 5% of opaque cells formed filaments under the same conditions. These results establish that C. albicans opaque cells do not form filaments efficiently under many of the standard in vitro conditions that induce white cell filamentation.

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Figure 1. Conditions inducing white cell filamentation do not induce opaque cell filamentation.

Environmental cues that induce efficient filamentation in C. albicans white cells (RBY717) include serum (A), high temperature (B), Spider medium (C and F), neutral pH medium (D), and Lee's medium (E). None of these environmental cues induce filamentous growth in opaque cells. Cell photographs were taken after 5 hours incubation at 37°C except for YPD supplemented with serum (2 hour incubation). Colonies grown on Spider medium were incubated at 30°C for 4 days.

https://doi.org/10.1371/journal.ppat.1003210.g001

Opaque cells undergo efficient filamentation in response to unique environmental cues

We next screened a series of in vitro culture conditions to identify environments that induce robust filamentous growth in opaque cells. Opaque-locked strains (overexpressing WOR1) or natural opaque strains were cultured on a variety of media, and colony and cell morphologies examined for evidence of filamentation. At least three distinct environmental cues were found to activate a program of opaque cell filamentation.

First, growth of opaque cells on medium containing the sugar sorbitol (synthetic complete dextrose medium supplemented with 1 M sorbitol; SOR medium) produced highly wrinkled colonies containing cells that were highly filamentous (Figure 2A). Filamentation was induced within 24 hours, and colony and cell morphologies are shown at 4 days of growth at 25°C. Staining of the cell walls with calcofluor white revealed that filaments consisted of cells with parallel sides and no constrictions at the septa, similar to true hyphae formed by white cells (Figure 2A).

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Figure 2. Novel environmental cues induce filamentation in C. albicans opaque cells.

Several culture conditions are described that induce filamentous growth in opaque-locked a cells (CAY2903). These include growth on (A) sorbitol (SOR), (B) minimal (MIN), (C) low nitrogen (SLAD), (D) low phosphate (LP) or (E) N-acetyl glucosamine (GlcNAc) medium at 25°C. These culture conditions do not induce filamentation in white a cells (RBY717). Panels show colony morphologies from mixed white/opaque populations (solid arrow, white colony; dashed arrow, opaque colony). Additional panels show white cells, opaque cells, and calcofluor white (CW)-strained opaque cells.

https://doi.org/10.1371/journal.ppat.1003210.g002

Second, growth on minimal medium (MIN medium; synthetic medium which lacks amino acids) induced filamentous growth producing highly wrinkled colonies (Figure 2B). Colony phenotypes were even more marked in minimal medium lacking nitrogen (SLAD medium) or minimal medium containing low phosphate concentrations (LP medium), as these conditions induced extensive peripheral filamentation around the edges of the colonies (Figure 2C and D). Filamentation was induced within 24 hours and colonies are shown after 4 days of growth at 25°C. Filaments from MIN, SLAD, and LP resembled pseudohyphae rather than true hyphae, as cells were highly elongated and branched, with slight constrictions at each of the branch points (Figure 2B–D).

Third, growth of opaque cells in the presence of the carbon source N-acetyl glucosamine (GlcNAc medium; Lee's medium containing 1.25% GlcNAc) induced efficient filamentation. Filamenting opaque cells resembled pseudohyphal cells with constrictions present at the septa (Figure 2E, cells shown after 24 hours and colonies after 4 days at 25°C). GlcNAc and SLAD have previously been reported to be activators of hyphal growth in white cells [37], [38], but we found that this required extended incubation for 5 days or longer at 37°C. In fact, white cells did not undergo filamentation under any of the conditions that efficiently induce opaque cell filamentation (compare white and opaque cell and colony morphologies in Figure 2).

Filamentous phenotypes were similar when using either natural opaque cells or those locked into the opaque state by constitutive WOR1 expression (compare Figure 2 and Figure S1). In addition, removal of cells from the filamenting opaque colonies gave rise to regular opaque colonies when incubated on SCD medium (data not shown). This result indicates that opaque cells did not switch to the white state when grown under filament-inducing conditions but were stably maintained as opaque cells.

The results described above were achieved using culture on solid media, but filamentation was also observed in liquid culture. For example, growth of opaque cells in liquid SOR or LP medium was found to efficiently induce filamentous growth (Figure S2B and C). In contrast filamentation in MIN or SLAD media was considerably reduced compared to that on solid media (Figure S2D and E). We therefore establish that opaque cells can filament in response to different environmental cues, and can do so when grown both in liquid culture and on agar plates.

The structure of filamentous opaque cells was further analyzed by scanning electron microscopy and compared to that of hyphal white cells (Figure 3). These images confirmed that opaque cells grown on SOR medium resembled hyphal cells (parallel sides with no constrictions) while those grown on LP medium resembled pseudohyphal cells (constrictions between elongated buds). These results establish that opaque cells can undergo a program of filamentous growth including either pseudohyphal-like or hyphal-like cells. Furthermore, we show that the environmental cues regulating filamentation in white and opaque cells are distinct, with different cues inducing filamentous growth in the two phenotypic states.

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Figure 3. Scanning electron micrographs of yeast and filamentous forms of C. albicans white and opaque cells.

(A) White cells (RBY717) were induced to form hyphae by growth in YPD supplemented with serum at 37°C. (B) Opaque cells (CAY2903) were induced to undergo filamentous growth by culture on LP or SOR media at 25°C. Scale bar, 5 µm.

https://doi.org/10.1371/journal.ppat.1003210.g003

Differential regulation of white/opaque filamentation by temperature

The yeast-hyphal switch in C. albicans white cells is strongly influenced by temperature, with most filamentation-inducing conditions occurring at elevated temperatures (e.g. serum induction of hyphae is increased at 37°C compared to 30°C [8]), although white cells also undergo filamentation at 25°C in response to certain cues [39], [40]. To determine if opaque cell filamentation is temperature-dependent, filament-inducing conditions were tested at 25°C, 30°C, and 37°C. Opaque filamentation was most efficient at 25°C, with decreased filamentation observed at 30°C, and even less filamentation at 37°C (Figure 4). We confirmed that the lack of filamentation at 37°C was not due to opaque cells switching back to white by using opaque-locked strains that have constitutive WOR1 expression. The thermal regulation of filamentation was similar when compared for SOR, MIN, SLAD and LP media, indicating that elevated temperatures generally inhibit opaque cell filamentation (Figure 4B–E). Again, white cells did not filament efficiently under any of the tested media conditions even when incubated at 37°C (Figure 4A–E), further demonstrating the specificity of the program of opaque filamentation.

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Figure 4. Differential thermal regulation of filamentous growth in white and opaque cells.

White (RBY717) and opaque (CAY2903) cells were cultured on SCD or filamentation-inducing media (SOR, MIN, SLAD, or LP) at 25°, 30°, or 37°C for 4 days. Opaque filamentation was optimal in the order 25°C > 30°C > 37°C. In contrast, white cell filamentation increased as temperatures increased, as evidenced by weak filamentation of white colonies on SCD medium at 37°C (top panels). (A) SCD, (B) SOR, (C), MIN, (D), SLAD, or (E) LP medium.

https://doi.org/10.1371/journal.ppat.1003210.g004

The heat shock protein Hsp90 has been implicated as a key regulator of temperature sensing in C. albicans. Elevated temperatures compromise Hsp90's functional capacity and this is thought to promote filamentation of white cells [8]. Pharmacological inhibition of Hsp90 (e.g. using the drug geldanamycin) therefore stimulates filamentation in white cells [8]. We used geldanamycin (GdA) to test if loss of Hsp90 also affects filamentation in opaque cells. White and opaque cultures were treated with geldanamycin in liquid YPD medium at 25°C and 30°C. Inhibition of Hsp90 caused efficient induction of filamentation in white cells at 30°C, but only a very limited morphological response in opaque cells (Figure S3). In fact, opaque cells were more susceptible to geldanamycin treatment than white cells, with the drug causing cell death in a large proportion of the population. Surviving opaque cells exhibited mixed morphologies, indicating that Hsp90 does not play as dominant a role in regulating opaque cell filamentation as it does in white cells.

The role of MAPK and cAMP signal transduction pathways in opaque filamentation

Multiple signaling pathways regulate filamentation in C. albicans white cells, including the MAPK pathway and the cAMP pathway that mediate filamentation in response to serum, temperature, CO₂, and starvation [2], [5]. The C. albicans MAPK pathway consists of a series of conserved kinases including Cst20, Hst7, and Cek1 that are homologous to S. cerevisiae Ste20, Ste7, and Kss1, respectively. The terminal Cek1 kinase activates Cph1, a transcription factor (homologous to ScSte12) responsible for inducing filamentous growth [6]. The cAMP pathway is similarly activated in response to multiple environmental cues and mediates filamentation via the transcription factor, Efg1 [7]. Together, Cph1 and Efg1 can be regarded as master regulators of C. albicans filamentation, and mutants lacking both Cph1 and Efg1 are highly defective in filamentation in white cells [41].

To evaluate the role of the MAPK and cAMP pathways in opaque filamentation, we constructed MTLa/a Δcph1/Δcph1 and Δefg1/Δefg1 mutants to analyze MAPK and cAMP signaling, respectively. Construction in an MTL homozygous strain was necessary to allow for strains to switch between white and opaque. Loss of Cph1 had little, if any, effect on opaque filamentation under any of the inducing conditions (SOR, MIN, LP, or GlcNAc media), indicating that the MAPK pathway does not play a major role in opaque filamentation. As shown in Figure 5, opaque Δcph1/Δcph1 colonies appeared similar to wildtype colonies; they exhibited extensive peripheral filamentation and consisted of pseudohyphal or hyphal cells, depending on the inducing medium. We also found that deletion of CPH1 had only a very modest effect on filamentation phenotypes in white cells (either MTLa/α or MTLa/a strains, see Figure S4), indicating that the MAPK pathway plays a relatively minor role in regulating filamentation compared to that of the cAMP pathway, consistent with previous observations [41].

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Figure 5. Analysis of the role of the cAMP and MAPK signaling pathways in opaque filamentation.

MTLa strains lacking EFG1 or CPH1 transcription factors that mediate the cAMP or MAPK signaling pathway, respectively, were analyzed for their ability to undergo opaque cell filamentation. Loss of EFG1 resulted in a defect in opaque filamentation under all conditions tested (right panels), while loss of CPH1 did not have a significant effect on opaque filamentation (left panels). Strains were cultured on SOR (A), SLAD (B) or LP (C) medium for 4 days at 25°C (colonies) or 22 hours at 25°C (cells). EFG1 mutants were CAY3526 (white) and CAY3292 (opaque), CPH1 mutants were CAY3524 (white) and CAY3298 (opaque), and the EFG1 complemented strain was CAY4384 (opaque).

https://doi.org/10.1371/journal.ppat.1003210.g005

The role of EFG1 in white and opaque cell filamentation was also addressed. We note that loss of EFG1 has been shown to increase switching from white to opaque [42]–[44], and that switching in efg1 mutants is regulated by pH [45]. We observed distinct white and opaque efg1 colonies on SOR, SLAD, and LP media, and loss of Efg1 resulted in decreased opaque cell filamentation under each of these culture conditions. For example, Δefg1/Δefg1 mutants were unable to form hyphal-like cells when grown on SOR medium and instead grew as chains of opaque cells (Figure 5A). Similarly, whereas wildtype opaque cells formed highly elongated pseudohyphae when grown on SLAD or LP media, efg1 mutants formed chains of normal looking opaque cells on these media (Figure 5B and C). Δefg1/Δefg1 mutants also produced smoother colonies compared to filamentous wildtype colonies (Figure 5A–C). Reintegration of the EFG1 gene into the mutant background restored filamentation, confirming that the mutant phenotype was due to the loss of this gene (Figure 5A–C). Loss of Efg1 was also shown to inhibit filamentation in white MTLa cells (Figure S4), consistent with its role in white a/α cells [7]. These results establish that Efg1 is a master regulator of filamentation in both C. albicans white and opaque cells.

The role of negative transcriptional regulators in white and opaque filamentation

Several negative regulators of filamentation play prominent roles in controlling the yeast-hyphal transition in white cells. These include the transcription factors Nrg1 and Rfg1, as well as the global repressor of gene transcription, Tup1. Loss of any one of these regulators results in white cells growing as hyphae or pseudohyphae under conditions that normally support yeast cell growth [36], [46]–[49]. Somewhat paradoxically, rfg1 mutants display a defect in hyphae formation under nutrient-limiting conditions [48], while overexpression of RFG1 promotes pseudohyphal growth [50]. We constructed mutants in each of these factors in switching-competent (MTL homozygous) strains to define their role in opaque filamentation.

As shown in Figure 6, loss of Nrg1 or Tup1 resulted in filamentation of opaque cells even when cultured on SCD medium, a medium that normally does not induce filamentous growth. Wor1 was constitutively expressed in these cells to drive formation of opaque cells, as tup1 mutants do not undergo stochastic switching to the opaque state, at least in the WO-1 strain background [51], [52]. Opaque colonies were extremely wrinkled in nrg1 and tup1 mutant strains, and consisted mostly of elongated pseudohyphal-like cells with constrictions at sites of cell division (Figure 6A and B). Since the highly filamentous phenotype of the tup1 mutant is similar between white and opaque cells, we also constructed fluorescence reporters to confirm the phenotypic state of these cells. pOP4-GFP and pWh11-mCherry constructs were employed to indicate the opaque or white state, respectively. As expected, the pOP4-GFP signal was higher in opaque tup1 cells than in white cells, although basal OP4 expression was evident in white cells (Figure S5B), consistent with previous observations [51]. Conversely, the pWh11-mCherry reporter was expressed in white cells but not opaque cells (Figure S5A). These results establish that opaque tup1 mutants are maintained in the opaque state and are as filamentous as white tup1 mutants.

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Figure 6. Negative transcriptional regulators of filamentation in white and opaque cells.

The roles of the transcription factors Nrg1, Tup1, and Rfg1, which are negative regulators of white cell filamentation, were tested in the opaque program of filamentous growth. Loss of Tup1 (CAY1616, opaque; CAY1550, white) or Nrg1 (CAY1618, opaque; CAY1552, white) resulted in white and opaque cell filamentation even when cells were grown on SCD medium (A and B). In contrast, loss of Rfg1 (CAY3299, opaque; CAY 3528, white) resulted in decreased filamentation on SOR (C), SLAD (D), and LP (E) medium. Loss of both CPH1 and RFG1 genes (CAY3151, opaque) resulted in a similar phenotype to rfg1 mutants (C–E). Loss of both EFG1 and RFG1 (CAY2822, opaque; CAY2701 white) generated phenotypes similar to efg1 and rfg1 single mutants (C–E). Colonies were grown on the different media for 5 days at 25°C and imaged.

https://doi.org/10.1371/journal.ppat.1003210.g006

In contrast to Nrg1 and Tup1, loss of Rfg1 reduced filamentation in opaque cells under the conditions tested. For example, growth of rfg1 mutants on SOR, SLAD or LP media produced very weak filamentation compared to the wildtype strain (Figure 6C–E). In general, rfg1 mutants grew as chains of opaque cells but cell shape was no longer extremely elongated and was reminiscent of the phenotype of efg1 mutants (compare Figure 6C–E and Figure 5A–C). We further examined the role of Rfg1 in the context of Cph1 and Efg1 that act in the MAPK and cAMP pathways, respectively. Opaque Δcph1/Δcph1 Δrfg1/Δrfg1 double mutants exhibited a similar phenotype to rfg1 mutants, indicating that Cph1 has no effect on opaque filamentation in the rfg1 background (Figure 6C–E). In addition, opaque efg1/rfg1 double mutants were analyzed, as both efg1 and rfg1 mutants exhibit reduced filamentation. It appeared that efg1/rfg1 opaque cells resembled efg1 and rfg1 single mutants, growing as chains of cells, and that some filamentation was still evident even in the absence of both transcription factors (Figure 6C–E).

Together, these results establish roles for Nrg1, Tup1, and Rfg1 in both white and opaque filamentation programs. Loss of Nrg1 or Tup1 transcriptional repressors results in activation of the program of filamentous growth in white and opaque cells. However, whereas Rfg1 is both a positive and negative regulator of filamentation in white cells depending on the conditions, we observe only a positive role for Rfg1 in promoting opaque cell filamentation.

Analysis of the Ume6-Hgc1 pathway in opaque filamentation

Recent studies have uncovered Ume6 as a key transcriptional regulator of hyphal and pseudohyphal growth in C. albicans white cells. In particular, it was shown that high levels of UME6 expression drive hyphal formation (and increase virulence), whereas intermediate levels of UME6 expression resulted in pseudohyphal growth [29]. The cyclin-related protein Hgc1 is an important downstream target of Ume6 and mediates agar invasion, hyphal extension, and formation of true septa [53]. Hgc1 functions acts as part of a Hgc1/Cdc28 complex that promotes filamentation by phosphorylating Rga2, a Cdc42 GAP protein, which in turn activates Cdc42 and drives actin polymerization [54]. In addition, both UME6 and HGC1 are negatively regulated by the Nrg1 and Tup1 transcription factors discussed above [25], [55]. Mutants in UME6 and HGC1 were constructed and tested in MTLa/a strain backgrounds to determine their contribution to opaque filamentation phenotypes.

Deletion of UME6 resulted in a marked decrease in opaque cell filamentation under each of the tested media conditions. Thus, growth of opaque ume6 mutants on SOR, LP, or MIN media generated chains of cells but cells were no longer highly elongated and no hyphal-like cells were observed. In fact, opaque ume6 cells resembled those of efg1 and rfg1 mutants (compare Figure 7 and Figure 6C–D). These results show strong parallels between white and opaque cells, as deletion of UME6 also compromises white cell filamentation [25], [55].

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Figure 7. Role of the Ume6-Hgc1 pathway in opaque cell filamentation.

The transcription factor Ume6 and the G1 cyclin-related protein Hgc1 were analyzed for their potential role(s) in filamentation in opaque cells. Mutant or complemented opaque strains were incubated on SOR (A and E), SLAD (B), LP (C and F), or MIN (D) medium for 4 days at 25°C and colony morphologies determined. Cell morphologies were photographed after 25 hours incubation. Loss of Ume6 (CAY1571) or Hgc1 (CAY4193) compromised opaque filamentation under each of the tested conditions, and filamentation was restored in the complemented strains CAY3697 (UME6 addback) and CAY4197 (HGC1 addback), respectively. Strain DK340 was the white ume6 mutant used.

https://doi.org/10.1371/journal.ppat.1003210.g007

In white cells, UME6 overexpression also drives cells into the hyphal form [29]. We therefore addressed whether UME6 overexpression is sufficient to induce opaque cell filamentation by using a strain in which UME6 is under the control of the Escherichia coli tet operator (tetO). The constructed strain also expresses an E. coli tet repressor-Saccharomyces cerevisiae Hap4 activation domain fusion protein. As a result, the UME6 gene is turned off in the presence of doxycycline, but is highly induced in the absence of doxycycline [29]. We found that overexpression of UME6 induced filamentous growth in both white and opaque cells, establishing UME6 as a master regulator of filamentation in both phenotypic states (Figure S6).

The role of HGC1 in opaque cell filamentation was also examined. These mutants were constructed in the P37005 background of C. albicans that is a natural MTLa/a isolate. The wildtype P37005 opaque cells underwent filamentation in response to inducing conditions, including on SOR and LP media. In contrast, P37005 Δhgc1/Δhgc1 mutants showed a marked defect in opaque filamentation, as these strains were unable to form filaments when grown on SOR or LP media (Figure 7E and F). The defect in hgc1 mutant filamentation was restored by complementation with the wildtype HGC1 gene (Figure 7E and F). Together, these results indicate a shared role for UME6 and HGC1 in promoting filamentation in both white and opaque cells.

Analysis of additional white cell filamentation regulators for roles in opaque cell filamentation

The Ras1 protein plays a prominent role in white cell filamentation and acts upstream of both the MAPK and cAMP pathways. Mutants in ras1 show a severe defect in hyphal growth in white cells under multiple conditions, while strains expressing a dominant active Ras1 mutation (Ras1^G13V) show enhanced hyphal formation [56]. Here, we tested the phenotype of Δras1/Δras1 mutants in opaque cells and observed a defect in opaque filamentation on SOR and LP media (Figure S7), consistent with cAMP signaling being necessary for filamentous growth (compare to the efg1 mutant phenotype, Figure 5). Curiously, expression of the constitutively active Ras1^G13V allele also partially suppressed filamentation on LP and SOR media (Figure S7). Thus, either loss of RAS1 function or hyperactive Ras1 activity appears to decrease filamentation of opaque cells.

Other pathways that regulate white cell filamentation include the Cph2/Tec1 pathway. Both of these factors are transcription factors and Cph2 is necessary for Tec1 expression, which in turn upregulates genes involved in hyphal development [57], [58]. Mutants in cph2 and tec1 were examined in the opaque phase but these mutants had no significant effect on filamentous growth under any of the tested conditions (Figure S8).

White cells are also induced to undergo filamentation when embedded in soft agar, and this program is dependent on the Czf1 transcription factor [59]. Opaque cells similarly underwent increased filamentation when cultured under embedded conditions (data not shown). Embedded conditions therefore represent an environmental cue that is conducive to inducing filamentation in both white and opaque cells. Interestingly, embedded growth is also one of the few filament-inducing conditions that is effective at 25°C for white cells [59]. As Czf1 promotes white filamentation under embedded conditions, opaque czf1 mutants were analyzed. This required overexpression of WOR1 to generate opaque cells, as czf1 mutants exhibit very low rates of white-to-opaque switching [44]. Opaque czf1 mutants produced an unusual ‘hyper-branching’ phenotype when grown on the surface on several media, including SLAD, LP and SOR medium (Figure S9). czf1 mutant opaque cells grew as highly branched chains that were clearly distinguishable from all other filamentation phenotypes. Czf1 is a therefore a regulator of filamentation in opaque cells, even when grown under non-embedded conditions.

Transcriptional profiling of filamentous opaque cells

Gene expression of filamentous white cells has been defined during growth in serum medium at 37°C [36], [60]. We set out to similarly define the transcriptional profile(s) of filamentous opaque cells, and to compare these profiles to those of filamentous white cells. Two medium conditions were used to induce opaque cell filamentation: low phosphate (LP) medium at 12 hours and sorbitol (SOR) medium at 16 hours (both at 25°C), as robust filamentation was observed at these time points. Expression was compared to that in SCD medium (non-filament inducing). We also compared gene expression profiles between white and opaque cells under these culture conditions.

First of all, we note that the master transcriptional regulators of the phenotypic switch were differentially expressed between white and opaque phenotypes under these culture conditions. Thus, WOR1, WOR2, and CZF1 were expressed at higher levels in opaque cells, while EFG1 was expressed at a higher level in white cells (data not shown). These results establish that cells are exhibiting the classical opaque transcriptional pattern when cultured in LP or SOR medium.

Comparison of filamenting and non-filamenting opaque cells revealed that 1188 genes were differentially expressed (by SAM) between LP and SCD medium, while 341 genes were differentially regulated between SOR and SCD medium. More specifically, 445 genes were induced and 743 genes repressed (>3-fold) in LP medium, while 143 genes were induced and 198 genes repressed in SOR medium. Presumably, these expression changes include many genes that are regulated by the nutritional change, as well as genes that directly mediate the transition from yeast to filamentous growth.

Comparison of gene expression profiles revealed that a core set of 48 genes was induced during filamentous growth of opaque cells in both SOR and LP medium (see Table S2). These genes were compared to those induced during white cell filamentation in serum at 37°C (Figure 8). In general, overlap between white and opaque filamentation profiles was limited, with most genes specific to one program or the other. Thus, many of the genes characteristic of hyphal formation in white cells, including ALS3, HYR1, PHR1, and SAP5, were not induced in filamentous opaque cells (Figure 8B). In fact, of the 55 genes induced in white cell hyphae, only 11 were induced in filamentous opaque cells in LP and SOR media. Significantly, one gene that was highly induced during both white and opaque filamentation was the key transcriptional regulator UME6. This result is consistent with the genetic requirement for this factor for filamentous growth in both white and opaque cells.

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Figure 8. Transcriptional profiling of opaque filamentation.

MTLa white (RBY717) or opaque (CAY2903) strains were incubated in SCD medium, a non-filament inducing condition, or sorbitol (SOR) and low phosphate (LP) media, conditions that specifically induce filamentation of opaque cells but not white cells. Cells were harvested after 12 hours growth (LP medium) or 16 hours growth (SOR medium) and cDNA prepared and hybridized against custom C. albicans ORF microarrays. (A) Comparative expression of opaque cell filamentation genes and white hyphal genes. Columns 1 and 2 represent opaque filamentation genes in LP and SOR media (relative to SCD controls) that passed SAM filters. Columns 3 and 4 represent hyphal-induced genes in white cells (serum-treated cells at 1 or 2 h compared to untreated cells) using data from Kadosh and Johnson [36]. Top panel; genes induced in both opaque and white filaments. Bottom panel, genes induced in opaque filaments but not white filaments. Grey boxes indicate no data available. (B) Genes expressed in white hyphal cells but not expressed in filamenting opaque cells. Top panel; genes induced in white hyphal cells only. Bottom panel, genes induced in white hyphal cells and either in opaque LP filaments or SOR filaments (but not both). op, opaque cells; wh, white cells. Gene numbers refer to orf19 gene identifiers (e.g. 1774 refers to orf19.1774).

https://doi.org/10.1371/journal.ppat.1003210.g008

In general, most hyphal-specific genes in white cells were not induced in filamenting opaque cells, and conversely opaque cells expressed filamentation genes not induced in white cells. In fact, several genes induced during opaque filamentation were repressed in white hyphal cells, including MNN22, OSM2, PCK1, and SAP98 (Figure 8B), while data for a number of opaque filamentation genes was not present in the white expression data set. Several opaque-specific filamentation genes are of interest, including PGA30 and PGA31 that encode putative GPI-anchored cell wall proteins. Upregulation of these genes suggests that filamentous opaque cells could exhibit altered adherence compared to yeast cells. A similar phenomenon has been observed in white cells, where expression of hyphal-specific surface proteins (e.g. ALS3, ALS10, ECE1, and HWP1) mediates increased adhesion of hyphae to host cells and promotes biofilm formation [61], [62]. We note that HWP1 expression was increased in both white and opaque filamentous cells (Figure 8B) and was also induced in opaque cells forming polarized mating projections [16], consistent with the model that HWP1 expression is directly regulated by actin dynamics during the morphological transition from yeast to polarized growth [63].

Together, these analyses reveal that the gene expression profiles of filamentous white and opaque cells are distinct, with only a limited number of factors induced in both programs of filamentation. However, the induction of UME6 in both gene profiles is consistent with a key role for this transcription factor in regulating filamentation in both white and opaque cell types.

Differential regulation of filamentous growth in C. albicans white and opaque cells

Our results demonstrate that the white-opaque switch plays a key role in regulating the yeast-hyphal transition in C. albicans. Thus, two of the best-studied programs regulating morphogenesis in C. albicans are interconnected, indicating overlap of the regulatory mechanisms involved in these programs. We show that opaque cells can form filamentous cells and do so in response to different environmental cues than those that induce filamentous growth in white cells. Thus, whereas serum, neutral pH, nutrient deprivation and high temperature are signals that induce filamentation in white cells, these stimuli do not induce filamentation in opaque cells (Figure 1). In contrast, opaque cells undergo filamentation in response to distinct cues, including sorbitol or low phosphate medium that do not induce filamentous growth in white cells (Figure 2). These results establish that white and opaque states are differentially programmed with respect to the integration of environmental signals for filamentous growth.

Genetic regulation of opaque filamentation

The regulation of filamentous growth in C. albicans white cells has been the subject of extensive studies (reviewed in [2], [5]). We examined whether the regulatory pathways controlling white cell filamentation, including MAPK and cAMP pathways, also function to regulate opaque cell filamentation. We show that Efg1, the terminal transcription factor in the cAMP pathway, and Ume6, a transcription factor that acts downstream of Efg1 [29], [53], [55], are key regulators of filamentation in opaque cells. Furthermore, overexpression of UME6 was sufficient to induce opaque cell filamentation, as it is in white cells [29]. We also observed roles for the positive regulator Hgc1 and the negative regulators Nrg1-Tup1 in opaque cell filamentation, similar to their established roles in white cell filamentation. Filamentous growth in white cells can be either positively or negatively regulated by Rfg1 depending on the conditions [48], and we found that rfg1 mutants were defective for filamentation in opaque cells. Thus, this transcription factor also plays a role in regulating filamentation in both white and opaque cells.

We also examined several other regulators of white cell filamentation for potential roles in opaque cell filamentation. Cph1, the master regulator of the MAPK pathway, did not influence opaque cell filamentation and we also observed that this factor has only a subtle role in white cell filamentation in the wildtype strain background. Similarly, Cph2 and Tec1 did not affect opaque filamentation, while deletion of Czf1 resulted in a hyper-branching phenotype specifically in opaque cells at 25°C. Taken together, our findings indicate that white and opaque filamentation occurs in response to different environmental stimuli, and generates different transcriptional responses (discussed below), but that genetic regulation of these programs involves many of the same signaling pathways in both phenotypic states (see model in Figure 9).

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Figure 9. Regulation of filamentous growth in C. albicans white and opaque cells.

The central programs regulating filamentation in white cells are shown, including the two core pathways of MAPK and cAMP-PKA signaling. White cell filamentation is also negatively regulated by the transcription factors Tup1 and Nrg1, and positively regulated by Ume6 in concert with the cyclin-like protein Hgc1. Filamentation in opaque cells is regulated by many of the same signaling pathways. In particular, Efg1 and Ume6 appear to be master regulators of filamentous growth in both white and opaque cells. However, there was no detectable role for MAPK signaling through Cph1, or for signaling via Cph2/Tec1, under the conditions used for inducing opaque filamentation.

https://doi.org/10.1371/journal.ppat.1003210.g009

Contrasting temperature regulation of filamentation in white and opaque cells

Filamentous growth in white cells is often dependent upon elevated temperatures, with several hyphal-inducing conditions requiring a temperature of 37°C for efficient filamentation [2], [5]. We found that filamentous growth in opaque cells exhibited the opposite dependence on temperature; opaque cells underwent filamentation efficiently at 25°C while filamentation was reduced at 30°C or 37°C. This was not due to opaque cells switching back to the white form at 37°C, as opaque cells were stably maintained by constitutive expression of WOR1, the master regulator of the opaque state [33]–[35]. In white cells, the molecular chaperone Hsp90 has recently been implicated in thermal regulation of the yeast-hyphal transition, as raised temperatures compromised Hsp90-mediated repression of hyphal formation [8]. In contrast to white cells, we found that inhibition of Hsp90 failed to elicit a large-scale change in morphology, although this could be due, at least in part, to the increased sensitivity of opaque cells to geldanamycin. These results reveal that elevated temperatures and/or decreased Hsp90 activity typically promote hyphal growth in white cells, while lower temperatures promote filamentation in opaque cells. Our findings have direct implications for pathogenicity by white/opaque cells, as they are likely to filament in different niches in response to different environmental cues, as discussed below.

Polarized growth and opaque filamentation

While this study revealed a novel program of filamentous growth in opaque cells, it is noted that polarized growth in opaque cells is not a new phenomenon. Opaque cells are the mating-competent form of C. albicans and respond to pheromones by forming mating projections that can be many times the length of the original opaque cell [16], [17]. Furthermore, opaque mating projections contain a Spitzenkörper-like structure in which the myosin regulatory light chain protein, Mlc1, localizes to a characteristic ball at the growing tip of the cell [18], [64]. The fact that opaque cells can form a Spitzenkörper indicates that these cell types undergo highly polarized growth by a mechanism similar to that in truly filamentous fungi. The discovery of environmental conditions that induces opaque filamentation now opens the door for further exploration of the regulation of opaque filamentation.

Comparison of white and opaque gene expression during filamentation

Gene expression profiles of filamenting white and opaque cells were distinct, with only 11 genes induced in both programs, while a further 44 genes were induced specifically in white hyphal cells and 37 genes induced only in opaque filamentous cells (Figure 8). One gene that was highly induced in both white and opaque states was the transcription factor UME6. As discussed above, UME6 is a critical regulator of filamentous growth in opaque cells, similar to its established role in white cells, and expression profiling confirms that this gene is co-regulated by morphogenesis in both cell types.

Interestingly, comparison of white-opaque regulated genes reveals that several regulators of filamentation are differentially expressed between white and opaque cells. For example, UME6, HGC1, NRG1, and EFG1, are white-opaque regulated genes; the first two genes are expressed at significantly higher levels in opaque cells, while the latter two genes show elevated expression in white cells (see Figure S10). EFG1 is itself a master regulator both of the white-opaque phenotypic switch and of filamentous growth [3], [43], [44]. It is therefore apparent that the transcriptional regulation of white/opaque phenotypes and that of filamentous growth are highly integrated, as previously suggested [65]. Presumably, the differential expression of these key regulators is at least partially responsible for the different propensities of white and opaque cells to filament in response to different environmental cues.

The role of white and opaque filamentation during infection

The yeast-hyphal transition is critical for infection by C. albicans white cells, where hyphae are more adherent and invasive than yeast-form cells. Despite this, questions remain as to the exact role of the hyphal structure during infection, and whether it is the genes that are co-regulated with the morphological switch that are critical for virulence or the hyphal structure itself [2]. In contrast, little is known about the potential for opaque cell filaments to promote pathogenesis. Early studies indicated that opaque cells are more effective at causing skin infections than white cells, and that opaque filamentation was induced on human skin epithelium [11], [12], [15]. Mating between opaque cells has also been shown to occur in this niche, indicating that the skin may represent a natural site for opaque colonization [66].

It is also possible that opaque filamentation occurs in other environmental niches, including those not associated with colonization and infection of the mammalian host. Future studies will examine both in vitro and in vivo conditions to determine those capable of inducing the program of filamentation in opaque cells. It is therefore an open question as to the role of the filamentous program in C. albicans opaque cells in infection and disease, and whether this program parallels that in white cells in promoting tissue destruction and pathogenesis.