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Vol. 19, Issue 3, 957-970, March 2008

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The Same Receptor, G Protein, and Mitogen-activated Protein Kinase Pathway Activate Different Downstream Regulators in the Alternative White and Opaque Pheromone Responses of Candida albicans

Department of Biology, The University of Iowa, Iowa City, IA 52242

Submitted July 20, 2007; Revised November 28, 2007; Accepted December 17, 2007
Monitoring Editor: Patrick Brennwald

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Candida albicans must undergo a switch from white to opaque to mate. Opaque cells then release mating type-specific pheromones that induce mating responses in opaque cells. Uniquely in C. albicans, the same pheromones induce mating-incompetent white cells to become cohesive, form an adhesive basal layer of cells on a surface, and then generate a thicker biofilm that, in vitro, facilitates mating between minority opaque cells. Through mutant analysis, it is demonstrated that the pathways regulating the white and opaque cell responses to the same pheromone share the same upstream components, including receptors, heterotrimeric G protein, and mitogen-activated protein kinase cascade, but they use different downstream transcription factors that regulate the expression of genes specific to the alternative responses. This configuration, although common in higher, multicellular systems, is not common in fungi, and it has not been reported in Saccharomyces cerevisiae. The implications in the evolution of multicellularity in higher eukaryotes are discussed.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Both single cell organisms and the individual cells of multicellular organisms must respond to a variety of environmental signals (Dorsky et al., 2000; Balázsi and Oltvai, 2005). In addition, in higher eukaryotes different cell types frequently respond to the same signal in unique ways (Rincón and Pedraza-Alva, 2003; Bacci et al., 2005; Dailey et al., 2005). In most cases, different signals interact with unique surface receptors that activate different signal transduction pathways, as has been demonstrated in Saccharomyces cerevisiae (Leberer et al., 1997; Gustin et al., 1998; Madhani and Fink, 1998; Elion, 2000). The mitogen-activated protein (MAP) kinase pathways have evolved as highly efficient, multipurpose signal transduction systems. S. cerevisiae uses multiple MAP kinase pathways, each one for a distinct signaling system, including the mating process, the filamentation process, cell wall integrity, ascospore formation, and osmoregulation (Levin and Errede, 1995; Gustin et al., 1998; Saito and Tatebayashi, 2004; Chen and Thorner, 2007). Several of these pathways share a limited number of components, but all are presumed to use different receptors to elicit very different responses. In the mating process of S. cerevisiae, a cells release a-factor that interacts with the a-receptor on cells, and cells release -factor that interacts with the -receptor on a cells. These alternative signals are then transduced through the same heterotrimeric G protein to activate the same MAP kinase pathway, which in turn activates the same downstream regulators that elicit similar mating responses, including G1 arrest, polarization, and shmooing (Sprague et al., 1983; Bender and Sprague, 1986; Leberer et al., 1997). Other fungi, including Magnaporthe grisea (Dixon et al., 1999; Zhao et al., 2005b) Neurospora crassa (Li et al., 2005), and Cryptococcus neoformans (Davidson et al., 2003; Kraus et al., 2003; Bahn et al., 2005) also use MAP kinase pathways for a variety of responses (Kruppa and Calderone, 2006).

The pathogenic yeast Candida albicans also uses MAP kinase pathways in the mating process (Chen et al., 2002; Magee et al., 2002), filamentation (Liu et al., 1994; Csank et al., 1998; Navarro-Garcia et al., 1998), and osmoregulation (Alonso-Monge et al., 1999; Smith et al., 2004). But C. albicans has one additional and unique response to mating pheromones that so far has not been identified in other yeast (Daniels et al., 2006). To mate, MTL-heterozygous strains of C. albicans must undergo homozygosis to a/a or / (Hull et al., 2000; Magee and Magee, 2000), then switch from the mating-incompetent white phenotype to the mating-competent opaque phenotype (Miller and Johnson, 2002; Lockhart et al., 2003a). Although pheromones induce mating responses in opaque a/a and / cells, including G1 arrest, polarization, and shmooing, as in S. cerevisiae, they do not induce these responses in white cells (Bennett et al., 2003; Lockhart et al., 2003a,b; Zhao et al., 2005a; Daniels et al., 2006). They do, however, elicit in white cells a dramatic increase in cohesion, adhesion, and biofilm development (Daniels et al., 2006). These changes have been demonstrated, at least in vitro, to result in a thicker white cell biofilm that provides a protective environment for spontaneously arising opaque cells to undergo mating (Daniels et al., 2006; Soll and Daniels, 2007).

Although mutational studies using complementation of auxotrophic traits as an assay for mating indicated that the -receptor Ste2p; the MAP kinases Cek1p and Cek2p, homologues of S. cerevisiae Kss1p and Fus3p; and a key target transcription factor, Cph1p, the homologue of S. cerevisiae Ste12p, were necessary for mating in C. albicans a/a cells (Chen et al., 2002; Magee et al., 2002; Bennett et al., 2003), the receptors, signal transduction pathways, and downstream transcription factor(s) that mediate the unique white cell response to pheromone remained unknown.

There existed at least three possible scenarios for this pathway. First, the same receptor, G protein complex, MAP kinase pathway, and targeted transcription factor could mediate both the opaque cell mating response and the white response. Second, select components of the signal transduction pathway regulating the opaque pheromone response could be shared with the pathway regulating the white pheromone response. Third, completely different receptors and transduction pathways, with no overlap, could mediate the alternative opaque and white responses. To distinguish between these possible scenarios, we generated deletion derivatives in a natural a/a strain for components mediating the mating response, including the -pheromone receptor gene, STE2; the gene for the β-subunit of the heterotrimeric G protein, STE4; the genes for the MAP kinases, CEK1 and CEK2; the gene for the downstream trans-acting factor CPH1 (the S. cerevisiae STE12 homologue); and the gene for the downstream cyclin-dependent kinase inhibitor, FAR1. We also generated deletion derivatives in natural / strain for the a-pheromone receptor gene, STE3, and for FAR1. Mutant and complemented strains were analyzed for the pheromone response of opaque cells and the pheromone response of white cells.

Our results demonstrate that the pathways regulating the alternative responses in opaque and white cells to the same pheromone share the same receptor, heterotrimeric G protein, and MAP kinase cascade, but they do not share the same downstream transcription factor(s). This represents a configuration in which two different cell types, white and opaque, respond to the same signals, and use the same receptors, heterotrimeric G protein, and the same MAP kinase cascade, but different downstream transcription factors, Cph1p in the opaque cell response and an as-yet-unidentified transcription factor in the white cell response. This configuration, which has no analogous example in S. cerevisiae, is found in a variety of multicellular systems in which the same signal is transduced in different cell types by the same signal transduction pathway, but results in different cellular responses (Rincón and Pedraza-Alva, 2003). We argue that several aspects of the signaling system between opaque and white cells suggest that it may represent an antecedent to multicellularity in higher eukaryotes.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Strain Maintenance and Growth
Strains used in this study and their origins and genotypes are listed in Supplemental Table S1. Cells of the natural strains P37005 [GenBank] (a/a) (Lockhart et al., 2002), P57072 [GenBank] (/) (Lockhart et al., 2002), and WO-1 (/) (Slutsky et al., 1987), the derived mutants, and complemented strains were maintained at 25°C on agar containing modified Lee's medium (Bedell and Soll, 1979) or YPD medium (Sherman et al., 1986). For distinguishing between white and opaque phase sectors or colonies, colonies were grown on modified Lee's agar medium supplemented with 5 µg/ml phloxine B, which differentially stained opaque phase cells red (Anderson and Soll, 1987). Before use, white and opaque phase cells were verified microscopically for the unique differences in cell shape and vacuole formation (Anderson and Soll, 1987; Slutsky et al., 1987).

Generation of Null Mutants
In this study, the following null mutants were generated: ste2, ste4, cek1, cek2, cek1 cek2, cph1, and far1 in the natural a/a strain P37005 [GenBank] ; and ste3 in the natural / strain P57072. In addition, a far1 mutant was generated in the natural / strain WO-1. The recyclable flipper cassette from pSFS2A (Reuss et al., 2004), containing a dominant nourseothricin resistance marker (CaSAT1), was used to create all mutants. The plasmid pSFS2A was a generous gift from Joachim Morschhauser (The University of Würzburg, Germany). The 4.2-kb XhoI–SacII fragment SAT1-2A of the cassette was blunt-ended with T4 polymerase before its use in ligations to create the deletion cassettes.

All of the primers used to create gene deletions are provided in Supplemental Table S2. To obtain a homozygous mutant strain for a particular gene, deletion cassettes I and II were generated in a two-step disruption strategy. Deletion cassette I was constructed as follows: 5' and 3' flanking regions of each target gene were amplified by polymerase chain reaction (PCR) by using the primers provided in Supplemental Table S2. The 5' region and 3' region were then each digested by SmaI and ligated together using T4 ligase. The 5'-3' fusion product was amplified by PCR and subcloned into the pGEM-T Easy vector (Promega, Madison, WI). The SAT1-2A fragment was then inserted into the SmaI-digested, dephosphorylated plasmid. This plasmid was digested with SacI plus SphI to generate the deletion cassette, which was then used to transform C. albicans strain P37005 [GenBank] , P57072 [GenBank] , or WO-1 by electroporation (De Backer et al., 1999). For each gene, two independent transformants were confirmed as heterozygous by both PCR and Southern analysis. The heterozygotes were then subjected to a pop-out strategy in the maltose-containing medium YPM (1% yeast extract, 2% Bacto-peptone, and 2% maltose) to excise the CaSAT1 marker. Deletion cassette II was constructed in a similar manner. The new 5' and 3' flanking regions that contained sequences deleted in the first step were amplified by PCR, by using the primers noted for each gene in Supplemental Table S2. The resulting plasmid was digested with SacI and SphI, and it was used to transform the heterozygous mutant derivatives. Two independent null mutants were confirmed by both PCR and Southern analysis for each gene.

Mutant Complementation
Complementation was performed for the mutants ste2, ste3, ste4, cek1, cek2, cph1, and far1, generating ste2/STE2, ste3/ste3, ste4/STE4, cek1/CEK1, cek2/CEK2, cph1/CPH1, and far1/FAR1. The CaSAT1 marker was deleted by a pop-out protocol from each null mutant as described for heterozygous mutants. The 5' and 3' regions flanking the stop codon were amplified by PCR with the primers noted for each gene in Supplemental Table S2. The 5'-3' fusion product was amplified by PCR and subcloned into pGEM-T Easy (Promega). For complemented strains, a DNA fragment containing both green fluorescent protein (GFP) and CaSAT1 was amplified by PCR with the primers noted in Supplemental Table S2, using plasmid pK91.6 (Srikantha and Soll, unpublished data) as template. GFP was inserted into the plasmid for future experiments and not used in this study. The GFP–CaSAT1 fragment was digested with BamHI plus BglII, and it was ligated into the Bg1II- or BamHI-digested, dephosphorylated plasmid containing the 5'-3' fusion product of the gene. This plasmid contained the transformation module for targeting to the gene locus. The in-frame GFP-gene fusion was confirmed by sequencing. This plasmid was digested with XhoI or StuI and used for transformation into the null mutant of each gene. Transformants were verified by both PCR sequencing and Southern analysis.

Opaque Cell Shmooing and Mating
Opaque cells were grown in liquid modified Lee's medium in a rotary water bath shaker (250 rpm) at 25°C to early saturation phase (5 x 10⁷ cells/ml) (Lockhart et al., 2003b). Cells were then pelleted, resuspended at 10⁶ cells/ml in fresh medium containing 3 x 10^–6 M synthetic 13-mer -pheromone (Bennett et al., 2003; Panwar et al., 2003) and incubated at 25°C in a shaker (250 rpm). The 13-mer peptide (GFRLTNFGYFEPG), synthesized by Open Biosystems (Huntsville, AL), was dissolved in dimethyl sulfoxide (DMSO). In controls not treated with pheromone, equivalent amount of DMSO was added. Shmooing and conjugation tube growth were monitored microscopically. Cell concentration was also monitored over time. To test for a-pheromone–induced shmoo formation, a transwell assay was performed according to Daniels et al. (2006).

To test for mating (Lockhart et al., 2003a), opaque cells of an a/a or / mutant were grown to early saturation phase and mixed with an equal concentration of WO-1 (/) or P37005 [GenBank] (a/a) opaque cells, respectively, in liquid culture. The mating mixtures were incubated at 25°C in a rotary shaker (250 rpm), and they were monitored for fusants microscopically over a 48-h period.

White Cell Cohesion and Adhesion Assays
To test for -pheromone–induced cohesion according to the methods of Daniels et al. (2006), a/a white cells from a saturation phase culture (4 x 10⁸ cells/ml) of strain P37005 [GenBank] or mutant derivatives were resuspended in fresh medium at a concentration of 5 x 10⁷/ml. The medium was supplemented with the 13-mer synthetic -pheromone at a concentration of 3 x 10^–6 M. The culture was rotated at 250 rpm at 25°C. Samples were taken from the suspension culture after 6 h, and they were examined microscopically for cell aggregates. To test for a-pheromone–induced cohesion of white cells of strain P57072 [GenBank] and the mutant derivative ste3, a 50:50 mixture of opaque P37005 [GenBank] and WO-1 cells was added to a suspension of either white P57072 [GenBank] or ste3 cells so that the former-inducing mixture made up 1% of cells. Opaque P37005 [GenBank] cells (a/a), stimulated by opaque WO-1 (/) cells, released a-pheromone.

To test for -pheromone–induced adhesion of white cells of the natural a/a strain P37005 [GenBank] and its mutant derivatives to plastic, the methods of Daniels et al. (2006) were used. Two milliliters of cells (5 x 10⁷/ml) were incubated in a well of a Costar six-cluster well plate (Corning Life Sciences, Lowell, MA) in the presence of 3 x 10^–6 M synthetic 13-mer -pheromone. After 16 h at 25°C, the wells were gently washed with phosphate-buffered solution and photographed. Gray scale images were subsequently pseudocolored for clarity. Three hundred microliters of a 0.05% trypsin-EDTA solution (Invitrogen, Carlsbad, CA) was added to each well. After 15 min, the cells on the dish bottom were released into 300 µl of supplemental Lee's medium containing 10% calf serum, and the number of adhering cells was determined in a hemocytometer. To test for a-pheromone–induced adhesion of / strain P57072 [GenBank] and its mutant derivative ste3, 1% opaque cells of a/a strain P37005 [GenBank] and / strain WO-1 were added to the well culture. After 16 h at 25°C, adhesion was analyzed as described above.

Biofilm Thickness
Biofilm enhancement was quantitated in strain P37005 [GenBank] and mutant derivatives according to a protocol described previously (Daniels et al., 2006), with one exception. Although in earlier experiments, a minority mixture (50:50) of opaque a/a and / cells was found more stimulatory than opaque / cells alone in enhancing a majority (90%) white a/a cell biofilm formation, recent experiments proved that minority opaque / cells (WO-1) alone induced near maximum enhancement of majority white biofilms. Therefore, a mixture of 90% white a/a test cells and 10% opaque / WO-1 cells (a total of 5 x 10⁷ cells in 2.5 ml of RPMI 1640 medium) was distributed on a silicone elastomer square in a well and incubated for 90 min. To test for enhancement of white cell biofilms of strain P57072 [GenBank] and the mutant derivative ste3, opaque a/a cells (P37005 [GenBank] ) (10%) were added to majority / cells in the presence of 3 x 10^–6 -pheromone. The square was then rinsed and incubated in RPMI 1640 medium on a rocker at 29°C for the subsequent 48 h. Biofilms were prepared in triplicate cultures. The biofilm was fixed, stained with calcofluor, and the thickness was measured using Bio-Rad LaserSharp software in a Bio-Rad Radiance 2100 MP laser scanning confocal microscope (LSCM) (Bio-Rad, Hermel, Hamstead, United Kingdom).

Quantitative Fluorescence Analysis of DNA
Two methods were used. In the first method, described in detail in Zhao et al. (2005a), opaque cells were grown to saturation phase, and then they were resuspended in fresh medium at 10⁶ cells/ml. Cells were treated with synthetic -pheromone (13-mer) in suspension, and then they were fixed after 3 h in 70% ethanol and treated overnight with RNase. Nuclei were stained with 25 µM Sytox Green (Invitrogen). Fluorescent quantitation of the staining of individual nuclei was performed using a confocal method we described in detail previously (Zhao et al., 2005a). Using the projected confocal image, a line profile of pixel intensity was measured across the center of each nucleus. In both control P37005 [GenBank] and far1 cell populations, only the nuclei of cells that had formed shmoos were scanned. That represented 60–70% of the P37005 [GenBank] cell population and 25% of the far1 cell population. In a second method, cell cycle status was determined by fluorescence-activated cell sorting (FACS). Cells were prepared as described above, with modification. RNase treatment was followed by proteinase K digestion. The final cell suspension was then stained overnight with 1 µM Sytox Green. The cells were sonicated briefly to disrupt cell aggregates, and then they were analyzed with a FACScan (BD Biosciences, Mountain View, CA). Cell cycle status was analyzed using ModFitLT version 2.0 software (BD Biosciences).

Northern and Southern Analyses
Northern and Southern analyses were performed as described previously (Lockhart et al., 2003b; Srikantha et al., 2006). For northern analyses, cells from saturation phase cultures were diluted into fresh medium in the absence or presence of 3 x 10^–6 M -pheromone, and they were pelleted after 4 h. Total RNA was extracted using the RNeasy Mini kit (QIAGEN, Valencia, CA). PCR products were used for probing Northern and Southern blots. The primers used to generate the PCR probe for each gene are listed in Supplemental Table S2.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The Pheromone Response Pathway Plays No Role in Switching
The deletion mutants ste2, ste4, cek1, cek2, cek1 cek2, cph1, and far1 were generated in the natural a/a strain P37005 [GenBank] (Lockhart et al., 2002). The deletion mutant ste3 (/) was generated in the natural / strain P57072 [GenBank] (Lockhart et al., 2002) and a far1 deletion mutant was generated in the natural / strain WO-1 (Slutsky et al., 1987). Each mutant and complemented derivatives were individually tested for spontaneous white-opaque switching by plating cells from single white colonies at low density on nutrient agar containing phloxine B, which differentially stained opaque cells red (Anderson and Soll, 1987). One thousand derivative colonies were scored for each strain. In every case, red colonies and/or sectors formed after 7 d at low frequencies similar to wild type (10^–3 opaque colonies). Cells from every tested red colony or sector of each mutant were found to exhibit the unique elongate opaque cell shape (data not shown). When these opaque cells were in turn plated at low density on agar, they formed a majority of opaque colonies and a minority of white colonies, demonstrating reversibility for every mutant. The same was true for the complemented strains ste2/STE2, ste3/STE3, ste4/STE4, cek1/CEK1, cek2/CEK2, cph1/CPH1, and far1/FAR1 (a/a). Zordan et al. (2006) demonstrated previously that switching was unimpaired in deletion mutants of STE2, CEK2, and FAR1 generated in an a cell background in a laboratory strain derived from strain SC5314. Together, these results demonstrate that the genes in the pheromone response pathway are not essential for white-opaque switching. This allowed ready isolation of white and opaque cells for each mutant and complemented strain, which were then tested for the alternative pheromone responses.

Opaque Cell Pheromone Response of ste2, ste3, and ste4
No shmoo formation was observed in opaque cells of parent strain P37005 [GenBank] in the absence of -pheromone. Seventy-five percent formed shmoos after 4 h of treatment with -pheromone and 91% after 8 h (Figure 1, A, B, and M). Neither opaque cells of the ste2 mutant nor of the ste4 mutant formed shmoos in response to -pheromone (Figure 1, C and D, respectively; and M). The complemented strains ste2/STE2 and ste4/STE4 regained the capacity to form shmoos in response to pheromone, and to the same extent as parental P37005 [GenBank] cells (data not shown). Bennett et al. (2003) also found that STE2 was required for opaque a cells to undergo shmoo formation.

View larger version (61K): [in this window] [in a new window]   Figure 1. -Pheromone does not induce conjugation tube ("shmoo") formation in opaque cells of the mutants ste2, ste4, the cek1 cek2 double mutant, or cph1, derived from the natural a/a strain P37005. (A and B) Representative images of P37005 cells in the absence (–) or presence (+) of -pheromone, respectively. (C, D, I, and J) Representative images of mutants ste2, ste4, cek1 cek2, and cph1, which did not form shmoos after 4 h of pheromone treatment. The same was true after 8 h (data not shown). Selected images of shmoo formation after 4 and 8 h for mutants cek1 (E and F), cek2 (G and H), and far1 (K and L). It should be noted that in these cases, the proportion of cells that had shmooed ranged between 9 and 68%; therefore, the images were selected. (M) Quantitation of shmooing for different strains. The "percentage of reduction in induced shmoos" was computed by dividing the difference in percentage of shmooing between P37005 and mutant strain, by percentage of shmooing of P37005, and multiplying by 100%. Bar (A), 5 µm.

View larger version (118K): [in this window] [in a new window]   Figure 2. a-Pheromone does not induce conjugation tube formation in opaque cells of the mutant ste3, derived from the / strain P57072. (A) Transwell apparatus for a-pheromone induction of / cells. (B) Representative image of opaque P57072 cells after 7 h of incubation in the response well. (C) Representative image of ste3 after 7 h in response well. Bar (C), 5 µm.

View larger version (87K): [in this window] [in a new window]   Figure 3. Opaque cells of the mutants ste2, ste3, ste4, the double mutant cek1 cek2, and cph1 do not mate (i.e., undergo fusion) with opaque cells of opposite mating type in suspension cultures, whereas mutants cek1, cek2, and far1 mate, but at reduced frequency. (A and D) Selected image of mating opaque cells of parent strain P37005 (a/a) with opaque cells of strain WO-1 (/), and P57072 (/) with P37005 (a/a), respectively. Selected images of mixtures of opaque cells of ste2 (B), ste4 (C), ste3 (E), the double mutant cek1 cek2 (H), and cph1 (I), with mating partners, none of which mated. Selected images of mixtures of opaque cells of cek1 (F), cek2 (G), and far1 (J), with mating partners, which underwent mating. (K) Quantitation of mating efficiency. "Percentage of reduction in mating" was computed as follows. The percentage of opaque cells of the different strains that fused with opaque cells of opposite mating type was subtracted from the percentage of the parent strain that fused with opaque cells of opposite mating type. The difference was then divided by the percentage of parent strain cells that fused, and the fraction multiplied by 100%. The percentage of mating cells of the complemented strains ste2/STE2, ste3/STE3, ste4/STE4, cek1/CEK1, cek2/CEK2, cph1/CPH1, and far1/FAR1 was similar to that of the parent wild type strains from which they were derived (data not shown). Bar (A), 5 µm.

White Cell Pheromone Response of ste2, ste3, and ste4
White cells of the mutants ste2 and ste4 were compared with white cells of the parental strain P37005 [GenBank] for the -pheromone–stimulated white cell response, which included dramatic increases in cohesion, adhesion, and enhanced biofilm development (Daniels et al., 2006). To assess pheromone-induced cohesion, ste2 and ste4 cells were incubated in suspension either in the absence or in the presence of -pheromone for 6 h. Cells were then distributed on a slide, and the average number of cells per aggregate was calculated. As we described previously (Daniels et al., 2006), the majority of white P37005 [GenBank] cells remained largely separated or formed small aggregates in the absence of -pheromone (Figure 4I), but in the presence of -pheromone, the majority of cells formed large aggregates (Figure 4, A and I). In the absence (data not shown) or presence of -pheromone, the majority of white ste2 and ste4 cells remained largely separated or formed small aggregates (Figure 4, B and I, and 4, C and I, respectively). White cells of the complemented strains ste2/STE2 and ste4/STE4 regained the aggregation response to -pheromone (data not shown).

View larger version (65K): [in this window] [in a new window]   Figure 4. White cells of the mutants ste2, ste4, and cek1 cek2 do not form large aggregates in response to pheromone as do wild-type cells, but mutants cph1 and far1 do. White cells of cek1 and cek2 form clumps of intermediate size. Cells of each strain from saturation phase cultures were diluted into fresh medium in the absence of pheromone (–) or in the presence of 3 x 10–6 M -pheromone (+). Samples were incubated 6 h before analysis. (A–H) Representative images of cells from parent and mutant strains in the presence of pheromone. (I) Average number of cells (error bar represents standard deviation) in aggregates. In total, 20 cell aggregates were analyzed for each strain. Bar (A), 5 µm.

View larger version (57K): [in this window] [in a new window]   Figure 5. In response to pheromone, white cells of the mutants ste2, ste4, and the double mutant cek1 cek2 do not form an adhesive film on the bottom of a plastic well. White cells of the individual mutants cek1 and cek2 form films nearly as dense as wild type, and white cells of the mutants cph1 and far1 form normal films. Dish bottoms were examined for a cell film after 16 h. Pseudocolor images (in orange) are provided. (A and B) Representative images of the dish bottom of P37005 cultures in the absence (–) and presence (+) of pheromone. (C–I). Representative images of the dish bottom of mutant cultures in the presence (+) of -pheromone. (J) Quantitation of cells adhering to the dish bottom. The "number of adherent cells" has been computed for the entire bottom of three separate wells. The average number is presented. Bar represents standard error. Data are also presented in J for complemented strains ste2/STE2, ste4/STE4, cek1/CEK1, and cek2/CEK2.

View larger version (89K): [in this window] [in a new window]   Figure 6. The thickness of biofilms formed by white cells of mutants ste2, ste4, and cek1, cek2, and the double mutant cek1 cek2, was not enhanced by minority (10%) opaque cells of opposite mating types, as was the thickness of the biofilms of parent strain P37005, cph1 and far1. A mixture of 90% white test cells and 10% opaque / cells was inoculated onto a silicone square and incubated for 48 h. The z-series projections were viewed from the side (90° tilt) of stacked multiphoton laser scanning confocal microscope scans. (A–L). Representative z-series projections of parental, mutant and complemented strain biofilms. (M) Average thickness (±standard deviation) of biofilms, computed from 10 measurements that included multiple cultures. Arrow span A represents 75 µm. Enhancement returned in the complemented strains ste2/STE2, ste4/STE4, cek1/CEK1, and cek2/CEK2.

To test whether a-pheromone induced ste3 cells to form a tight adhesive film on a plastic surface, majority white cells (99%) of either strain P57072 [GenBank] or strain ste3, were mixed with a minority (1%) of half opaque P37005 [GenBank] (a/a) cells and opaque WO-1 (/) cells. As in the previous strategy, opaque / cells stimulate the release of a-pheromone by opaque a/a cells, which then stimulates white-specific responses in white / cells (Daniels et al., 2006). Whereas white P57072 [GenBank] cells were induced to form an adhesive film on the plastic well bottom, ste3 cells were not (Supplemental Figure S2). Pheromone-induced substrate adhesion was restored in the complemented strain ste3/STE3 (Supplemental Figure S2). Minority opaque a/a cells (P37005 [GenBank] ) stimulated by -pheromone also enhanced biofilm formation by majority white P57072 [GenBank] cells, but not majority white ste3 cells (Supplemental Figure S3). The former were close to twice as thick as the latter (Supplemental Figure S3). Enhancement of biofilm formation by minority opaque cells similar to that in parent strain P57072 [GenBank] was restored in the complemented strain ste3/STE3 (Supplemental Figure S3). Together, these results demonstrate that the same a-pheromone receptor (Ste3p) regulates the a-pheromone–induced opaque cell response and white cell response, just as the same Ste2p receptor regulates the -pheromone responses of opaque and white a/a cells.

Opaque Cell Pheromone Response of cek1, cek2, and the Double Mutant
Opaque cells of the individual MAP kinase mutants cek1 and cek2, which were generated in the natural a/a strain P37005 [GenBank] , formed shmoos in response to -pheromone, but the response in both cases was delayed, and the proportion of cells that responded after 4 h was reduced (Figure 1, E and G, respectively; and M). The percentage of cells that shmooed in both mutants increased after 8 h of treatment (Figure 1, F and H, respectively; and M). Opaque cells of both mutants also underwent mating with opaque cells of the / strain WO-1 (Figure 3, F and G, respectively), but the proportion of fusants was reduced by >99% for cek1 and by 90% for cek2 (Figure 3K). Pheromone-induced shmooing and mating of opaque cells was restored in the complemented strains cek1/CEK1 and cek2/CEK2 (data not shown).

However, opaque cells of the double mutant cek1 cek2 neither shmooed in response to -pheromone (Figure 1, I and M) nor underwent mating with opaque cells of the / strain WO-1 (Figure 3, H and K). These results were consistent with those obtained with null mutants of KSS1 and FUS3, the respective orthologues of CEK1 and CEK2, in S. cerevisiae (Elion et al., 1991), and they confirm and extend earlier observations by Chen et al. (2002) on cek1, cek2, and the cek1 cek2 double mutant of C. albicans strain CAI4, in which complementation was used between auxotrophic a and strains as an assay for mating.

White Cell Pheromone Responses of cek1, cek2, and the cek1 cek2 Double Mutant
In the absence of -pheromone, white cek1 and cek2 cells formed only small aggregates in suspension cultures (data not shown), as did white P37005 [GenBank] cells (Figure 4I). In response to -pheromone, both white cek1 and white cek2 cells formed aggregates that were, on average, larger than in the absence of -pheromone (Figure 4, D and E, respectively), but still far smaller than those formed by -pheromone–treated P37005 [GenBank] cells (Figure 4A). The average number of cells per clump for treated white cek1 and cek2 cells was 35 and 25, respectively, compared with 125 for white P37005 [GenBank] cells (Figure 4I). In response to -pheromone, white cek1 and white cek2 cells formed a film on a plastic surface (Figure 5, E and F, respectively). Quantitation revealed that the number of adherent cells in these films was greater than that in unstimulated cultures, but consistently smaller (p < 0.001) than that of pheromone-stimulated P37005 [GenBank] cells (Figure 5J). This adhesive response was regained in the complemented strains cek1/CEK1 and cek2/CEK2 (Figure 5J). A minority of / opaque cells of strain WO-1 did not enhance the thickness of biofilms formed by majority cek1 or cek2 white cells over a 48-h period (Figure 6, D and E, respectively), as it did white P37005 [GenBank] cells (Figure 6M). Enhancement of the thickness of white cell biofilms by minority / opaque cells returned in the complemented strains (Figure 6, K and L, respectively; and M).

In the presence of -pheromone, white cells of the double mutant cek1 cek2 formed only small clumps (Figure 4, F and I) like untreated cells (data not shown). White cek1 cek2 cells also did not form a film on a plastic surface in response to -pheromone (Figure 5, G and J). Finally, minority / opaque cells of strain WO-1 did not stimulate an increase in the thickness of a majority a/a white cell biofilm of the cek1cek2 mutant on a silicone elastomer surface (Figure 6, F and M). These results demonstrate that as was the case for STE2, STE3, and STE4, the partially redundant functions of CEK1 and CEK2 are necessary for both the opaque and white cell responses, suggesting that the response pathways from receptor through the MAP kinase cascade are shared.

Opaque Cell Pheromone Response of cph1
Opaque cells of the trans-acting factor mutant cph1, which was generated in the natural a/a strain P37005 [GenBank] , neither formed shmoos in response to -pheromone (Figure 1, J and M), nor mated with opaque cells of the / strain WO-1 (Figure 3, I and K). The complemented strain cph1/CPH1 reacquired these responses (data not shown). These results support and extend earlier observations by Magee et al. (2002) and Chen et al. (2002) in which complementation between auxotrophs was used as an assay to demonstrate that mating depends on CPH1 function.

White Cell Pheromone Response of cph1
Although deletion of CPH1 completely blocked pheromone-induced formation and mating of opaque cells, it did not block the white cell pheromone response. Treatment of white cph1 cells with -pheromone stimulated aggregation in suspension cultures (Figure 4, G and I) to levels comparable with that of treated white P37005 [GenBank] cells (Figure 4, A and I). Treatment with -pheromone also induced cph1 cells to form a tightly adhering film on a plastic surface (Figure 5, H and J) comparable with that formed by treated white P37005 [GenBank] cells (Figure 5, B and J). Finally, minority / opaque cells of strain WO-1 stimulated an approximate threefold increase in the thickness of majority white cph1 cell biofilms (Figure 6, G and M), an increase comparable with that induced by / cells in white P37005 [GenBank] biofilms (Figure 6, A and M). These results clearly demonstrate that the white cell response to pheromone does not require the downstream target Cph1p. Therefore, although the pheromone response pathway from receptor through the MAP kinase pathway is shared, the downstream components of the pathways regulated by the MAP kinases differ.

Opaque Cell Pheromone Response of far1
It was demonstrated previously that white cells do not shmoo (Bennett et al., 2003; Lockhart et al., 2003b) and that they do not arrest in G1 in response to -pheromone (Zhao et al., 2005a). Therefore, one might not expect FAR1 to play a role in the white cell response, because the role FAR1 plays in the analogous mating process of S. cerevisiae is in the polarization of cells in a gradient of pheromone and G1 arrest (Chang and Herskowitz, 1990; Valtz et al., 1995; Butty et al., 1998). However, in supplemental data to Roberts et al. (2000), it was reported that STE12 was not up-regulated by pheromone in a far1 mutant, indicating that Far1p played a role in the up-regulation of pheromone-induced genes. Therefore, we considered the possibility that Far1p may also be involved in the up-regulation of genes by pheromone in the white cell response. The far1 mutant of S. cerevisiae shmoos in response to -pheromone, but the shmoos do not polarize in a gradient of pheromone, and far1 cells are not blocked in G1 by pheromone (Chang and Herskowitz, 1990; Dorer et al., 1995; Valtz et al., 1995). The far1 mutant of S. cerevisiae is capable of mating, but the frequency of mating is significantly reduced, presumably because far1 cells cannot efficiently find partners because they are defective in chemotropism (Valtz et al., 1995).

Opaque cells of the C. albicans far1 mutant generated in the a/a strain P37005 [GenBank] , exhibited mating-associated abnormalities similar to those in S. cerevisiae. Opaque cells of C. albicans far1 shmooed in response to -pheromone (Figure 1, K and L), but the percentage of shmooing was reduced by 57% after 4 h and 81% after 8 h (Figure 1M). The C. albicans far1 mutant also exhibited a strong mating defect (99.26% reduction) compared with the parental strain P37005 [GenBank] (Figure 3, J and K). To test whether bilateral mating between far1 (a/a) and a far1 (/) strain completely blocked mating, we generated a far1 mutant in the / strain WO-1. More than 4000 cells were scanned in mixtures of opaque far1 (a/a) and far1 (/). No mating was observed (data not shown). These results suggest that FAR1 is not essential for shmooing in response to pheromone, although the frequency is reduced, but FAR1 seems to be essential for fusion. Full shmooing and mating responses were restored in the complemented C. albicans strain far1/FAR1 (data not shown).

To test whether FAR1 was required for a pheromone-induced block in G1 during shmoo formation in C. albicans, the DNA content of the nuclei of opaque cells of strain P37005 [GenBank] , the far1 derivative, and the complemented far1/FAR1 strain undergoing shmooing was assessed by measuring the pixel intensity of a line scan through the nucleus of cells stained with Sytox Green according to methods described previously (Zhao et al., 2005a). At saturation phase in liquid culture, the distributions of maximum pixel intensities of the stained nuclei of 27 independently scanned opaque cells of each of strain P37005 [GenBank] (Figure 7A), far1/FAR1 (Figure 7B), and far1 (Figure 7C), which in all three cases were primarily unbudded, were similar, ranging between 100 and 150 relative units. When opaque cells from saturation phase cultures of P37005 [GenBank] , far1/FAR1, or far1 were diluted into fresh medium and incubated for 3 h in the absence of -pheromone, the range of maximum pixel intensities in all cases increased. In the control P37005 [GenBank] or far1/FAR1, the range increased to 150–250 relative units, and in far1, to 110–250 relative units (data not shown), indicating a lag in far1 cells. If cells from the two control strains P37005 [GenBank] and far1/FAR1 were diluted into fresh medium containing -pheromone and incubated for 3 h, the increases in D