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. 2005 Jun;16(6):2903–2912. doi: 10.1091/mbc.E05-01-0073

Induction of the Candida albicans Filamentous Growth Program by Relief of Transcriptional Repression: A Genome-wide AnalysisD⃞

David Kadosh 1, Alexander D Johnson 1
Editor: Thomas Fox1
PMCID: PMC1142434  PMID: 15814840

Abstract

Candida albicans, the major human fungal pathogen, undergoes a reversible morphological transition from blastospores (round budding cells) to filaments (elongated cells attached end-to-end). This transition, which is induced upon exposure of C. albicans cells to a number of host conditions, including serum and body temperature (37°C), is required for virulence. Using whole-genome DNA microarray analysis, we describe 61 genes that are significantly induced (≥2-fold) during the blastospore to filament transition that takes place in response to exposure to serum and 37°C. We next show that approximately half of these genes are transcriptionally repressed in the blastospore state by three transcriptional repressors, Rfg1, Nrg1, and Tup1. We conclude that the relief of this transcriptional repression plays a key role in bringing the C. albicans filamentous growth program into play, and we describe the framework of this transcriptional circuit.

INTRODUCTION

The yeast Candida albicans is the major human fungal pathogen. Although normally present as a commensal organism in the human digestive tract, Candida can cause oral and vaginal thrush, as well as a variety of more serious mucosal and systemic infections. Unlike most pathogens, Candida is capable of infecting all the major organs and tissues of the human body and has no known reservoir outside of warm-blooded animals (Odds, 1988, 1994a, 1994b; Dupont, 1995; Weig et al., 1998). Candidiasis is now the fourth-leading cause of hospital-acquired infections in the world with an attributable mortality of up to 35% (Edmond et al., 1999). Immunocompromised individuals, such as cancer patients undergoing chemotherapy, AIDS patients, and organ transplant recipients are particularly susceptible to Candida infections (for reviews see Shepherd et al., 1985; Dupont, 1995; Weig et al., 1998). Currently, approximately $1 billion per year is spent on the treatment of patients with hospital-acquired infections in the United States alone (Miller et al., 2001).

Several properties of C. albicans are known to contribute to its virulence. These include adhesiveness to host cells, secretion of degradative enzymes, and—the subject of this article—the ability to undergo a reversible morphological transition from the blastospore (single-celled) to filamentous (elongated cells attached end-to-end) forms (Lo et al., 1997; Mitchell, 1998; Brown and Gow, 1999; Ernst, 2000a, 2000b; Calderone and Fonzi, 2001; Brown, 2002b; Calderone and Gow, 2002; Saville et al., 2003). The filamentous forms of C. albicans encompass two distinct morphologies, pseudohyphae and hyphae. In pseudohyphae, the cells are attached end-to-end but each cell has an elliptical shape with constrictions at the septa. In true hyphae, these constrictions are absent and a row of cells show a relatively uniform width. Pseudohyphae and hyphae also differ in other respects, including the precise ways that mitosis and cell division are carried out (Odds, 1985, 1988; Sudbery et al., 2004). In this article, we use the term filamentous to include both the pseudohyphal and hyphal forms. This term is useful because many of the same conditions that induce the hyphal form also induce the pseudohyphal form; indeed, mixed populations of pseudohyphae and hyphae are a common outcome of filament-inducing conditions (Odds, 1985, 1988; Brown, 2002a).

The blastospore-filament transition is accompanied by the induction of many genes. Although some of these genes are required for the morphological transition per se, others are not (Braun et al., 2000; Nantel et al., 2002). This latter class of genes presumably encode proteins that give the filamentous forms their distinctive properties. Although the blastospore-filament transition is clearly required for virulence (Lo et al., 1997; Braun et al., 2000; Braun et al., 2001; Murad et al., 2001; Saville et al., 2003), the relative importance of the morphological forms themselves, compared with the changes in gene expression that accompany them, is not known.

Candida undergoes the transition from blastospores to filaments in response to a wide variety of laboratory conditions, many of which mimic the environment of mammalian host tissues. These conditions include growth at elevated (body) temperature (37°C), at neutral pH or in the presence of serum or certain human hormones (Kinsman et al., 1988; Bramley et al., 1991; Caticha et al., 1992; Mitchell, 1998; Brown and Gow, 1999; Ernst, 2000a). One of the strongest sets of filament-inducing conditions is the combination of body temperature (37°C) and serum. These conditions trigger a signal transduction pathway that ultimately results in the activation of a complex transcriptional program important for C. albicans filamentous growth and virulence (for reviews, see Ernst, 2000b; Brown, 2002b).

In this article, we address the mechanism by which the filament-specific genes become active, and we focus on three key negative regulators, Rfg1, Nrg1, and Tup1. As previously described, strains missing any one of these regulators are highly attenuated for virulence and grow filamentously under non–filament-inducing conditions. Moreover, each of these deletion strains inappropriately expresses at least some of the filament-specific genes in the absence of any inducer (Braun and Johnson, 1997; Braun et al., 2000; Braun et al., 2001; Kadosh and Johnson, 2001; Khalaf and Zitomer, 2001; Murad et al., 2001). In the well-studied baker's yeast, Saccharomyces cerevisiae, Nrg1 and Rox1 (the homolog of Rfg1) are sequence-specific DNA-binding proteins and Tup1 is a global transcriptional repressor recruited to genes via interaction with promoter-specific DNA-binding proteins such as Rox1 and Nrg1 (Keleher et al., 1992; Deckert et al., 1995; Tzamarias and Struhl, 1995; Park et al., 1999; Smith and Johnson, 2000). Although not directly demonstrated, a similar relationship is very likely to hold for C. albicans Rfg1, Nrg1, and Tup1.

In this article, we address the following questions: what is the full spectrum of genes transcriptionally induced during the blastospore to filament transition? How much of this program is due to relief of negative control by Nrg1, Rfg1, and Tup1? What is the relationship (superimposing, overlapping, or distinct) between the Rfg1- and Nrg1-repressed genes? Do Rfg1 and Nrg1 act solely through Tup1 or do they have other mechanisms available to them? Are Nrg1, Rfg1, and Tup1 devoted exclusively to the regulation of the filamentous growth program or do they have other roles in the cell as well? To answer these questions, we utilized whole-genome microarrays to analyze the blastospore-filament transition, to identify all genes repressed by Rfg1, Nrg1, and Tup1 and to establish the functional relationships among these three transcriptional repressors.

MATERIALS AND METHODS

Strains

In all experiments, the wild-type strain used was CAF2-1 (Δura3;imm434/URA3; Fonzi and Irwin, 1993). All rfg1, nrg1, and tup1 strains used were homozygous deletion mutants and have been described previously (Braun and Johnson, 1997; Braun et al., 2001; Kadosh and Johnson, 2001). Where possible, multiple isolates of each mutant strain were used for experimental repetitions.

Media and Growth Conditions

In all experiments performed to compare gene expression in rfg1, nrg1, and tup1 mutant strains with the wild-type strain, cells were grown under standard noninducing conditions: yeast extract-peptone-dextrose (YEPD) medium at 30°C (Guthrie and Fink, 1991). For all strains, a saturated overnight culture was diluted to OD600 = 0.2 in 1 L of YEPD medium and allowed to grow at 30°C until OD600 ∼ 1.0, at which point cells were harvested for RNA preparation. In half of the nrg1 and tup1 experiments, a saturated overnight culture was diluted into 1 L of YEPD medium and grown at 30°C overnight to OD600 ∼ 1.0 on the following day; at this point cells were collected for RNA preparation.

In the serum- and temperature-induction experiments, a saturated overnight culture of the wild-type (CAF2-1) strain was diluted into 2 L of YEPD medium and allowed to grow at 30°C overnight until cells reached an OD600 of 12.6 (experiment 1) or 14.4 (experiment 2). An aliquot of this culture (the zero time point) was then harvested to prepare RNA and additional 200-ml aliquots were diluted into 2 L of fresh, prewarmed YEPD medium in the presence or absence of 10% fetal calf serum (FCS) and grown at 30 or 37°C. Cells were harvested for RNA preparation at 1-, 2-, 3-, and 5-h time points. The OD600 of the zero time point was critical for optimal filament induction (see Results section).

DNA Microarray Experiments and Analysis

C. albicans DNA microarray construction, preparation of RNA and cDNA, hybridization of coupled cDNA to microarrays and DNA microarray analysis were performed as described previously (Bennett et al., 2003). Data normalized by NOMAD (http://ucsf-nomad.sourceforge.net/) were filtered to include only spots showing a median signal intensity ≥500 in at least one channel. The rfg1 experiment was performed four times, and the nrg1 and tup1 experiments were done six times (all from independently grown cultures). In all four of the rfg1 experiments and in half (three) of the nrg1 and tup1 experiments, each hybridization contained a mutant sample (labeled with Cy5 dye) and wild-type sample (labeled with Cy3 dye). In half (three) of the nrg1 and tup1 experiments and in both serum and temperature induction experiments, each hybridization contained an experimental sample (labeled with Cy5 dye) and a universal reference sample (labeled with Cy3 dye) generated from cells grown under a wide variety of filament-inducing and noninducing conditions; later, the signal ratios (ratio of the median signal intensities of each spot) for each mutant sample versus reference were divided by the signal ratios for the wild-type sample versus reference and signal ratios for each time point of the serum and temperature-induction experiment versus reference were divided by the signal ratios for the zero time point versus reference. Signal ratio values for wild-type versus reference and the zero time point versus reference represented median values obtained from at least three independent DNA microarray experiments (performed using cDNA obtained from the same total RNA preparation). Significance analysis of microarrays (SAM) was performed as described previously (Tusher et al., 2001), using the One-class Response, K-nearest neighbors settings (10 neighbors) and the default Random Number Seed (1234567). For each data set, 600 permutations were carried out and delta values were selected to calculate low false discovery rates (FDRs) for the median d-scores. Gene annotation and analysis was based on the C. albicans orf19 assembly (Braun et al., 2005) and, for YHB and ALS family genes, on the Candida Genome Database (Arnaud et al., 2005). Please note that in the case of three SAP family genes (SAP4, SAP5, SAP6) and genes in the MRS512 (FGR6) family (including orf19.914, orf19.1099, orf19.5191, orf19.5775, orf19.7005), it was impossible to distinguish between family members by microarray experiments because the genes in these families were nearly identical at the DNA level. Gene classification was carried out based on the S. cerevisiae gene ontology (GO) terms (Dwight et al., 2002; Harris et al., 2004).

RESULTS

Optimization of Conditions for Induction of the Blastospore to Filament Transition in Response to Serum and Temperature

Northern analysis has previously shown that several filament-specific genes undergo a large (>20-fold) transcriptional induction when C. albicans is treated with serum at 37°C (Birse et al., 1993; Staab et al., 1996; Braun and Johnson, 2000). In our initial experiments to monitor the genome-wide response of C. albicans to serum and temperature, we observed much lower induction ratios than those cited above. These low induction ratios were also observed by Nantel et al. (2002), who described the first microarray analysis of the response of C. albicans to serum and temperature. In our experiments, we found that the low induction ratios were largely due to the presence of mixed populations of blastospores and filamentous forms in both the induced and noninduced cultures. This results in a significant underestimation of the extent of transcriptional induction and obscures genes that are only moderately induced. By experimenting with a variety of different protocols, we found that diluting an overnight culture 1:10 directly into fresh, prewarmed YEPD medium with 10% serum at 37°C gave optimal induction of filamentous growth in large cultures. For reasons we do not understand, the optical density of the overnight culture was critical. Cells induced from a culture at OD600 ∼ 10.6 or lower showed incomplete filament formation and a correspondingly reduced expression of filament-specific genes. In contrast, cells induced from a saturated overnight culture generated filaments even when diluted into noninducing medium (fresh YEPD medium at 30°C), a phenomenon that we term “hyphal shock.” Only when the optical density of the overnight culture was OD600 ∼ 13 did we observe 100% filament formation and consequently high levels of filament-specific gene expression under inducing conditions and little or no filament formation under noninducing conditions. We do not understand why this particular optical density is required for optimal induction, but it is possible that the nutrient availability, quorum sensing, or some other factor plays an important role in priming the cells for efficient serum and temperature induction.

Transcriptional Profile of Genes Induced During the Blastospore to Filament Transition in Response to Serum and Temperature

Using the conditions described above we carried out two independent serum- and temperature-induction experiments with a wild-type C. albicans strain (CAF2-1). Over a time course, we observed the response to serum alone, temperature alone (37°C), and serum plus temperature (37°C). For each condition, cells were grown in YEPD medium (plus serum, when appropriate) and harvested at 1-, 2-, 3-, and 5-h time points for RNA extraction and DNA microarray analysis. We also monitored cell morphologies microscopically at each time point. As shown in Figure 1, 100% of the cells were induced to form true hyphae when grown at 37°C in the presence of serum, and most cells remained in this form at the 5-h time point. Cells grown at 30°C in the presence of serum or at 37°C in the absence of serum also showed significant filament formation. However, under these conditions, the morphology change was less pronounced: not all of the cells were in filamentous form, particularly at the 5-h time point, and many of the filamentous cells (especially at 30°C in the presence of serum) were in the pseudohyphal rather than true hyphal form. Nearly all the cells grown in YEPD at 30°C were in the blastospore form for the duration of the experiment (Figure 1).

Figure 1.

Figure 1.

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Morphology of cells undergoing the blastospore to filament transition. An overnight culture grown in YEPD medium at 30°C was diluted 1:10 into prewarmed YEPD medium at 30°C or at 37°C in the presence or absence of 10% FCS. Induction time (hrs.) is shown on top. Aliquots of cells were fixed in 4.5% formaldehyde, washed twice with 1 × phosphate-buffered saline, and then visualized by Nomarski optics and photographed under 100× magnification. Please note that the 0-h time point photograph shows cells immediately before induction.

For the microarray experiments, the results from each time point were normalized to the median value (taken from three microarrays) of the 0 time point (30°C, no serum). We observed 61 genes transcriptionally induced ≥2-fold by serum and temperature (37°C) together when compared with the same time points in the 30°C cultures without serum. Approximately one-third of these genes were induced ≥10-fold (Table 1). The majority of the 61 genes were reproducibly induced in both time courses and at multiple time points within each time course. A few genes were induced only at the earliest time point (t = 1 h); for these genes we relied solely on the reproducibility between the two time courses to assign significance to them. We will refer to these 61 genes as hyphal-specific, because they are differently expressed in 37°C plus serum (where the cells are 100% hyphal) compared with 30°C without serum where the cells are >95% in the blastospore form (Figure 1). Of the 61 hyphal-specific genes, most showed a characteristic transcription profile in response to serum at 37°C, with the highest level of expression observed at the 1-h time point and a progressive decrease at later time points (Figure 2). By a similar analysis, we identified 25 genes whose expression is reduced in the presence of serum at 37°C; see Supplementary Data.

Table 1.

Serum- and temperature-induced genes

No. of these genes upa
No. of serum- and temperature-induced genesb >2-fold >5-fold >10-fold
61 61 33 22
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a

Number of genes showing the indicated mean fold induction at the 37°C + 10% serum 1-h time point (n = 2). Please note that a single late-induced gene, orf19.6079, shows a mean fold induction >2-fold at the 37°C + 10% serum 3-h time point

b

Criteria for selection: 1) mean induction ≥2-fold (n = 2) at the 37°C + 10% serum, 1-h time point, 2) mean of (37°C + 10% serum)/(30°C only) ratio >2.0 for the 1-h time point. Please note that a single late-induced gene, orf19.6079, is an exception to these criteria

Figure 2.

Figure 2.

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Transcriptional profile of most serum- and temperature-induced genes. Fold induction, relative to the zero time point, is shown for four serum- and temperature-induced genes (HYR1, ALS3, HWP1, SAP5) when cells were grown in YEPD medium, at 30 or 37°C in the presence or absence of 10% FCS, at the indicated time points. *Please note that SAP5 expression levels may reflect, in part, SAP4 or SAP6 expression levels due cross-hybridization on the microarray, because these gene family members are nearly identical at the DNA level.

When C. albicans was exposed to 37°C in the absence of serum or to serum at 30°C, the majority of the hyphal-specific genes were also induced, but the overall level of induction was lower than that observed for the serum-37°C combination (Figure 2). As described above, at most time points, the 37°C alone and serum alone cultures contained mixtures of blastospores, pseudohyphae, and hyphae. Our microarray results are most consistent with the hyphal-specific genes also being induced in pseudohyphal cells, but the lack of pure populations of pseudohyphae prevent a definitive conclusion. These results indicate that serum or temperature (37°C) alone can bring the filamentous transcription program into play, but that the combination does so more efficiently.

As noted above, Nantel et al. (2002) also monitored the response of C. albicans to serum and temperature using microarrays. They identified 18 genes induced ≥2-fold, with a single gene induced greater than 10-fold. Ten of the ≥2-fold induced genes are included in our list of 61 hyphal-specific genes. As described above, the growth conditions used in our study enabled us to obtain pure populations of blastospores and hyphae, and this clear separation brought the differentially expressed genes into sharp relief.

A Significant Fraction of Genes Induced by Serum and Temperature during the Blastospore to Filament Transition Are under Negative Control by Rfg1, Nrg1, and Tup1

Having identified a defined set of genes that is induced during the blastospore to hyphal transition in response to serum and temperature, we next determined the extent to which these genes are controlled by the Rfg1, Nrg1, and Tup1 repressors. As described in the Introduction, these transcriptional repressors are known to control aspects of filamentous growth as a deletion of any one of their genes leads to constitutive filamentous growth on noninducing medium (30°C without serum). To identify the genes controlled by these three repressors, we used C. albicans genome-wide arrays to compare the profiles of an isogenic set of strains, each strain deleted for the gene encoding one of the repressors, grown under noninducing conditions (30°C, no serum). As described in Materials and Methods, mRNA from each deletion strain was isolated from 4 to 6 independent cultures and compared with mRNA from an isogenic parental strain.

To identify the complete sets of target genes for each repressor, we turned to the significance analysis of microarrays (SAM) program, which ranks target genes by statistical significance (Tusher et al., 2001). Using low false discovery rates, we identified a set of 165 genes showing significantly elevated expression in the Δtup1/Δtup1 strain and sets of 124 genes each showing increased expression in Δrfg1/Δrfg1 and Δnrg1/Δnrg1 strains (Table 2; Supplementary Data). Many of the genes show low (<2-fold), but statistically significant effects, and probably represent secondary effects of deleting these repressors (see below).

Table 2.

Total number of genes showing significantly elevated expression in rfg1, nrg1, and tup1 mutants

Strain SAM false discovery rate (%)a No. of genes significantly up
rfg1 3.24 124
nrg1 0.78 124
tup1 0.15 165
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a

Significance analysis of microarrays (SAM) was used to define gene sets based on the indicated false discovery rates (FDRs). See Materials and Methods for details

To determine which hyphal-specific genes are controlled by Tup1, Nrg1, or Rfg1, we considered all genes induced ≥2-fold when Tup1, Rfg1, or Nrg1 was deleted and compared this to the list of 61 hyphal-specific genes. By these criteria, about half of the hyphal-specific genes are repressed by one or more of these negative regulators (Table 3). If we consider only hyphal-specific genes that are induced ≥5- or ≥10-fold, the fraction of genes controlled by these repressors is somewhat higher, approximately two-thirds (Figure 3; Supplementary Data). Thus, by any of these criteria the three repressors play a key role in orchestrating the filamentous growth program; they seem particularly important in regulating genes that show the highest hyphal-specific induction.

Table 3.

Fraction of serum- and temperature-induced genes controlled by Rfg1, Nrg1, and Tup1

No. of these genes also up ≥2-folda
No. of genes induced by 37°C + serumb rfg1 nrg1 tup1
61 17 (28) 26 (43) 30 (49)
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a

Median fold effects were determined from four (rfg1) or six (nrg1 and tup1) microarray experiments. Genes with less than two data points for any one mutant were excluded from the analysis. Percentage in parentheses

b

See Table 1 for selection criteria

Figure 3.

Figure 3.

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Cluster diagram of the top serum- and temperature-induced genes (≥5-fold mean induction, n = 2, at the 37°C + 10% FCS 1-h time point) with corresponding rfg1, nrg1, and tup1 mutant data. Only data from one serum- and temperature-induction experiment and only genes with greater than 89% of data present are shown. Each column represents data from a single microarray experiment. Red, increased expression; green, reduced expression; gray, no data available. *Please note that SAP5 expression levels may reflect, in part, SAP4 or SAP6 expression levels due cross-hybridization on the microarray, because these gene family members are nearly identical at the DNA level.

Functional Relationships among the Rfg1, Nrg1, and Tup1 Repressors

As described in the Introduction, Rfg1 and Nrg1 are sequence-specific DNA-binding proteins and Tup1 is a global repressor believed to be recruited by Rfg1, Nrg1, and other DNA-binding proteins. We used the results of the microarray analysis performed on strains deleted for each repressor to further test this idea and to establish the detailed relationships among the three repressors.

Previous microarray studies in S. cerevisiae have shown that when the Tup1 transcriptional repressor is deleted, both direct and indirect transcriptional effects can be observed (DeRisi et al., 1997; Green and Johnson, 2004). Typically the direct effects (genes directly controlled by Tup1) show larger magnitudes of change, although the correlation is not strict. To get a better idea of the relationships among the Rfg1, Nrg1, and Tup1 repressors we wished to minimize indirect effects. To this end, we considered genes showing a median increase in expression ≥5-fold in the deletion strain compared with wild type. By these criteria, Tup1 represses 103 genes; Nrg1 and Rfg1 repress 37 and 25 genes, respectively (Tables 4 and 5). Although these data sets are likely to be incomplete (compared with the SAM-based analysis described above), we believe they are enriched for direct effects and therefore optimal for addressing the question at hand.

Table 4.

Fraction of top Rfg1- and Nrg1-repressed genes controlled by Tup1

Strain Total no. of genes up ≥5-folda No. of these genes with tup1 mutant datab No. of these genes also up ≥2-fold in tup1 mutantc
rfg1 25 21 12 (57)
nrg1 37 31 29 (94)
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a

Median fold effects were determined from four (rfg1) or six (nrg1) microarray experiments (comparing the rfg1 and nrg1 strains to a wild-type strain). Only genes with two or more data points and the majority of data points ≥2-fold were included in the analysis

b

Indicates number of genes with two or more data points for the tup1 mutant, as determined from six microarray experiments

c

Percentage in parentheses

Table 5.

Fraction of top Tup1-repressed genes controlled by Rfg1 and Nrg1

Total no. of genes up ≥5-fold in tup1 mutanta No. of these genes with nrg1 mutant datab No. of these genes also up ≥2-fold in nrg1 mutantc No. of these genes with rfg1 mutant datab No. of these genes also up ≥2-fold in rfg1 mutantc
103 87 42 (48) 70 19 (27)
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a

Median fold effects were determined from six microarray experiments (comparing the tup1 strain with a wild-type strain). Only genes with two or more data points and the majority of data points ≥2-fold were included in the analysis

b

Indicates number of genes with two or more data points for the nrg1 or rfg1 mutants, as determined from four (rfg1) or six (nrg1) microarray experiments

c

Percentage in parentheses

Using this strategy, we observed the relationships between the three repressors. Approximately 60% of the genes derepressed ≥5-fold in the Δrfg1/Δrfg1 strain also show elevated expression in the Δtup1/Δtup1 mutant, suggesting that the majority of Rfg1 repression works via Tup1, but that a significant Tup1-independent pathway also exists (Table 4). Nrg1, in contrast, appears to function almost entirely via Tup1, because 94% of the top Nrg1-controlled genes also show increased expression in the Δtup1/Δtup1 mutant (Table 4 and Figure 4). The reciprocal relationship, that is the fraction of Tup1-controlled genes regulated by Rfg1 and Nrg1, was determined in the same way. Approximately half of the genes derepressed ≥5-fold in the Δtup1/Δtup1 mutant showed elevated expression in the Δnrg1/Δnrg1 strain and approximately one-quarter showed increased expression in the Δrfg1/Δrfg1 strain (Table 5). Many of the Rfg1-controlled genes in this set are also controlled by Nrg1, and we estimate that about half of the Tup1 repression observed in C. albicans occurs through Nrg1 and/or Rfg1; it is therefore likely that several additional sequence-specific DNA-binding proteins also function through Tup1.

Figure 4.

Figure 4.

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Cluster diagram of genes showing a median elevated expression ≥5-fold (n = 6) in the nrg1 mutant with corresponding tup1 mutant data (n = 6). Only genes with greater than 66% of data present are shown. Each column represents data from a single microarray experiment. Red, increased expression; gray, no data available.

Classes of Genes Induced by Serum and Temperature and Repressed by Nrg1, Rfg1, and Tup1

To systematically classify the serum- and temperature-induced and Rfg1-, Nrg1-, and Tup1-controlled target genes, we generated a list of gene classes based on universal gene ontology (GO) terms derived from S. cerevisiae (Dwight et al., 2002; Harris et al., 2004). Approximately 75% of target genes (depending on the gene set) were classified based on S. cerevisiae GO terms (see Supplementary Data for GO terms associated with each gene class). Genes lacking S. cerevisiae homologues or reciprocal hits were classified as either “unknown function” (∼18%) or “function in other organism” (∼7%). Using this system, we identified 25 gene classes, 21 of which describe the genes induced by serum and temperature. As shown in Figure 5, these genes function in a wide variety of biological processes, including ER/Golgi/secretion and the cell cycle.

Figure 5.

Figure 5.

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Representation of gene classes induced during the blastospore to filament transition compared with the genome as a whole. Asterisks indicate gene classes which are overrepresented in the serum- and temperature-induced gene set compared with the genome as a whole. Gene classification was based on S. cerevisiae gene ontology (GO) terms (Dwight et al., 2002; Harris et al., 2004). Please note that the “unknown function” gene class includes all genes of unknown function (with and without S. cerevisiae homologues or reciprocal hits).

We next determined whether any gene classes are overrepresented in the serum- and temperature-induced gene set compared with the genome as a whole, and we found four classes: cell wall components, cell motility/budding/cell division genes, secreted/degradative enzymes, and genes involved in ER/Golgi/secretion (Figure 5). The first class, cell wall components, is also overrepresented in all three mutant data sets and the second class, cell motility/budding/cell division genes, is overrepresented in both Nrg1 and Tup1 mutant data sets (see Supplementary Data). The implications of these overrepresentations are presented in the Discussion.

DISCUSSION

Induction of the C. albicans Filamentous Growth Program Involves Relief of Transcriptional Repression

Numerous transcriptional regulators have been shown to be important for controlling the transition from the blastospore form to the hyphal form of C. albicans (Ernst, 2000b; Brown, 2002b). Here, using whole-genome DNA microarray analysis, we have identified 61 hyphal-specific genes that are induced in response to growth in serum at 37°C, and we show that approximately half of these genes are under negative control by the Rfg1, Nrg1, and/or Tup1 transcriptional repressors (Figure 6). These results, in combination with previous findings (Braun and Johnson, 1997; Braun et al., 2001; Kadosh and Johnson, 2001; Khalaf and Zitomer, 2001; Murad et al., 2001), show that relief of negative regulation plays a key role in the transition from blastospores to hyphae.

Figure 6.

Figure 6.

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A model for control of genes induced during the blastospore to filament transition by the Rfg1, Nrg1, and Tup1 pathways. Rfg1, Nrg1, and at least one additional DNA-binding protein (X) bind to the promoters of filament- and virulence-specific target genes and direct transcriptional repression via recruitment of the Tup1 corepressor. The Nrg1 transcript itself has previously been shown to be down-regulated in the presence of 10% serum and temperature (Braun et al., 2001; Murad et al., 2001). Rfg1 repression activity is proposed to be regulated by filament-inducing conditions at the posttranslational level (Kadosh and Johnson, 2001). Target genes are indicated in boxed areas. *Cell wall components; +ER/Golgi/secretion genes.

We also observed the response of C. albicans blastospores to serum at 30°C and to 37°C in the absence of serum. The vast majority, if not all, of the hyphal-specific genes were also induced under these conditions, although the extent of induction of each gene was generally less than that observed for the 37°C plus serum conditions. Although 37°C plus serum gave rise to nearly 100% hyphal cells, 30°C plus serum and 37°C in the absence of serum produced a combination of blastospores, pseudohyphae and hyphae. The simplest interpretation of our result is that many, if not all, of the hyphal-specific genes are also induced in pseudohyphae. Thus, serum or 37°C can bring about the filamentous growth program, but the combination of serum and 37°C does so much more efficiently.

Specific Roles of Rfg1, Nrg1, and Tup1 in Regulating Filamentous Growth

By the criteria described in the results section, Nrg1 and Tup1 work together to repress 20 hyphal-specific genes (Figure 6). As described in the introduction, Nrg1 mRNA is down-regulated by serum and temperature (Braun et al., 2001; Murad et al., 2001); it is likely that the hyphal-specific genes controlled by Nrg1-Tup1 are induced as a direct result of this down-regulation. Rfg1 and Tup1 together repress 13 hyphal-specific genes, although it is not currently known how Rfg1 repression is lifted during the blastospore-hyphal transition. A significant number of hyphal-specific genes (10) are controlled by both Rfg1 and Nrg1, indicating some degree of overlap between these two pathways, whereas other hyphal-specific genes are controlled by only one of these two regulators. Finally, our microarray results predict the existence of at least one additional sequence-specific DNA-binding protein (unknown at this point) that recruits Tup1 and represses 7 hyphal-specific genes (Figure 6).

Although Rfg1, Nrg1, and Tup1 clearly have a key role in hyphal-specific gene regulation, our results also indicate that about half of the genes induced by serum and temperature are controlled independently of them. It is likely that direct transcriptional activation is responsible for much of this gene regulation, as a number of transcriptional activators have been implicated in the filamentous growth program (for reviews, see Ernst, 2000b; Brown, 2002b). We also note that in the case of some genes controlled by Rfg1-Tup1 or Nrg1-Tup1 (such as HYR1 and PHR1), the level of expression is often higher during filamentous growth than it is in a strain grown in noninducing conditions but deleted for the appropriate repressor. Thus, at least some of the hyphal-specific genes that are negatively controlled by Nrg1, Rfg1, and Tup1 are likely also to be under direct positive control during filamentous growth.

Genes Involved in a Wide Variety of Biological Processes Are Induced during Filamentous Growth by Relief of Transcriptional Repression

Many of the genes induced in the blastospore-hyphal transition made conceptual sense. For example, a high proportion of the hyphal-specific genes encode cell wall components (Figures 5 and 6), some of which are known to function as adhesins important for adherence to host cells (Staab et al., 1999; Zhao et al., 2004); it has been known for many years that hyphae and blastospores differ in their adherence properties (Odds, 1985, 1988). A number of these cell wall components, such as HWP1, ALS3, and HYR1 have been studied directly (Bailey et al., 1996; Hoyer et al., 1998; Staab et al., 1999); the role of others, such as PST1 and IHD1, have not been addressed experimentally. The majority of the cell wall class of proteins are under negative control by Rfg1, Nrg1, and Tup1 (Figure 6).

We also found that several of the hyphal-specific genes repressed by Rfg1, Nrg1, and Tup1 encoded proteins important for cell division. These genes included KIP4, a kinesin heavy-chain homolog (Hoyt et al., 1992); CDC10, a septin involved in cytokinesis (Warenda and Konopka, 2002); and RAX2, a homolog of a membrane protein involved in bipolar budding (Kang et al., 2004). It is well known that the mechanisms of cell division differ between blastospores, pseudohyphae, and hyphae (Sudbery et al., 2004), so this class of hyphal-specific genes makes intuitive sense. A number of hyphal-specific genes also encode secreted/degradative enzymes, such as members of the secreted aspartyl protease (SAP) family, which have long been known to play a role in C. albicans virulence (Hube, 1996; Hube and Naglik, 2002).

Several hyphal-specific genes controlled by Rfg1, Nrg1, and/or Tup1 appear to function in processes not previously implicated in filamentous growth and virulence. For example, CBP1 encodes a human corticosteroid-binding protein with a putative oxidoreductase activity (Malloy et al., 1993); this could be important in allowing hyphae to sense the local environment. DCC1 functions as a putative DNA damage checkpoint protein (Chang et al., 2002) that could be important for enabling C. albicans to cope with oxidative stress in the host environment. Two genes induced by serum and temperature (UME6 and USV1) encode putative transcriptional regulators (Tzung et al., 2001; Segal et al., 2003), and it is possible they are responsible for activating a subset of the hyphal-specific genes through a regulatory cascade.

We also note the presence of one additional hyphal-specific gene class, ER/Golgi/secretion, in which the majority of genes do not appear to be under negative control by Rfg1, Nrg1 or Tup1 (Figure 6). The role of this gene class in filamentous growth and virulence is less obvious, but may signal a general need for increased protein expression and secretion during the morphological change.

Finally, a significant number of hyphal-specific genes, some of which are under negative control by the repressors described in this study, encode proteins of unknown function (Figure 6). Most of these genes are unique to C. albicans (at least so far), suggesting they mediate specific aspects of commensalism or pathogenesis.

Relationship between Virulence and the Blastospore-Hyphal Transition

Of the 61 genes induced by serum and temperature, 7 are known to be important for virulence in a mouse model of systemic candidiasis to our knowledge (Ghannoum et al., 1995; Sanglard et al., 1997; Staab et al., 1999; Braun et al., 2000; Warenda et al., 2003; Martchenko et al., 2004); virulence studies of the remaining genes, at least to our knowledge, have not been reported. It is also known that mutations that affect the regulation of hyphal-specific genes also lead to attenuated virulence (Lo et al., 1997; Kadosh and Johnson, 2001; Saville et al., 2003). Thus the blastospore-hyphal transition, which includes the induction of the 61 genes identified here, is clearly an important aspect of C. albicans virulence. The work in this article shows the key importance of negative transcriptional control in regulating this process and identifies a large set of genes that are implicated in specific aspects of virulence.

Supplementary Material

[Supplemental Material]
mbc_E05-01-0073_index.html (2.5KB, html)

Acknowledgments

We thank Mike Lorenz, Gerald Fink, Joe DeRisi, and Diane Inglis for the collaboration that resulted in the production of our C. albicans DNA microarrays. We thank B. Braun for bioinformatics assistance, P. O'Farrell for use of the differential interference microscope in his laboratory, B. Braun, A. Uhl, and D. Inglis for C. albicans genome annotation efforts and members of the Johnson laboratory, past and present, for technical advice and fruitful discussions during the course of the experiments. We also thank S. Argimon and A.J.P. Brown for communicating results before publication. C. albicans sequence data were provided by the Stanford Genome Technology Center (http://www-sequence.stanford.edu/group/candida), supported by the NIDR and the Burroughs Wellcome Fund. The work presented in this paper was supported by a UCSF Biochemistry National Institutes of Health (NIH) postdoctoral training grant to D.K. and by grants from the Burroughs Wellcome Fund (993218) and NIH (R01 AI49187) to A.D.J.

This article was published online ahead of print in MBC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E05-01-0073) on April 6, 2005.

D⃞

The online version of this article contains supplemental material at MBC Online (http://www.molbiolcell.org).

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

[Supplemental Material]
mbc_E05-01-0073_index.html (2.5KB, html)
mbc_E05-01-0073_Data_Set_1.txt (4.3KB, txt)
mbc_E05-01-0073_Data_Set_2.xls (19KB, xls)
mbc_E05-01-0073_Data_Set_3_-_Complete_data_set.txt (3.1MB, txt)
mbc_E05-01-0073_Data_Set_4_-_Serum_and_Temp.-induced_genes.txt (29.4KB, txt)
mbc_E05-01-0073_Data_Set_5.txt (13.4KB, txt)
mbc_E05-01-0073_Data_Set_6_-_Complete_Tup1_up_genes.txt (18.3KB, txt)
mbc_E05-01-0073_Data_Set_7_-_Top_Tup1_up_genes.txt (37.3KB, txt)
mbc_E05-01-0073_Data_Set_8.txt (4.7KB, txt)
mbc_E05-01-0073_Data_Set_9_-_Complete_Nrg1_up_genes.txt (14.1KB, txt)
mbc_E05-01-0073_Data_Set_10_-_Top_Nrg1_up_genes.txt (11.1KB, txt)
mbc_E05-01-0073_Data_Set_11.txt (4.7KB, txt)
mbc_E05-01-0073_Data_Set_12_-_Complete_Rfg1_up_genes.txt (13.8KB, txt)
mbc_E05-01-0073_Data_Set_14.txt (4KB, txt)
mbc_E05-01-0073_Data_Set_13_-_Top_Rfg1_up_genes.txt (9.7KB, txt)

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