INTRODUCTION

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Chitin is a polymer of the Saccharomyces cerevisiae cell wall that has been shown to be essential for cell viability (8, 36). Chitin is assumed to confer strength to the cell wall, allowing cells to grow in nonstabilized media. In vivo, calcofluor, a fluorescent brightener that interacts specifically with chitin (30), acts as an antifungal agent by killing fungal cells (30, 31). This antifungal effect is associated with an increase in the rate of chitin synthesis, although the polymer synthesized is abnormal and does not confer enough strength on the cell wall to guarantee cell survival. Based on these observations, it has been assumed that chitin is the primary target of the drug, a hypothesis that was reinforced by the isolation of several mutants resistant to calcofluor, all of them showing severe reductions in the levels of chitin synthesis (32, 38). The first mutants resistant to calcofluor that do not have apparent defects in chitin synthesis have been described only very recently (19). However, the characterization of these mutants was preliminary, and from the report it is difficult to deduce the existence of other cellular targets for the action of calcofluor. The effect of calcofluor is also characterized by an increase in chitin synthesis that is dependent on chitin synthase III activity and on de novo protein synthesis (31). These results suggest that calcofluor induces some intracellular responses that are triggered by the drug binding to the cell wall chitin.

S. cerevisiae contains several mitogen-activated protein kinase (MAPK) cascades that translate physiological modifications into intracellular responses (see reference 11 for a recent review). The protein kinase C (PKC) cascade is involved in cell wall integrity and morphogenesis (17) by participating in the control of -glucan synthesis (29), although recently it has been shown that this pathway also plays a role in the transcriptional control of CHS1, CHS2, and CHS3, the genes for the catalytic subunits of chitin synthases (13). Despite this, the relationship between the PKC pathway and chitin synthesis is not clear because the regulation of chitin synthesis in S. cerevisiae mostly occurs by posttranslational modification of Chs3p (7, 9). In theory, a hitherto-uncharacterized relationship might exist between other MAPK pathways, i.e., those involved in mating or sporulation, and cell wall synthesis because S. cerevisiae requires cell wall reorganization at different stages of its biological cycle (8).

S. cerevisiae contains a fourth MAPK pathway, the high-osmolarity glycerol response (HOG) pathway. It is basically involved in the adaptation of cells to high-osmolarity environments and is required for S. cerevisiae growth in media supplemented with high NaCl (0.9 M) or sorbitol (1.5 M) concentrations (11). The HOG pathway is well known in yeast and has been shown to be made up of three typical kinases (MAPKKK, MAPKK, and MAPK), which are products of the SSK2-22, PBS2, and HOG1 genes, respectively (11). Activation of this route in response to high osmolarity can take place through two independent routes: a two-component regulatory sensor (SLK1-SLN1), which activates the first kinase (21), or a single sensor (SHO1), which transfers the signal to the PBS2 kinase (22) through the MAPKKK protein Ste11p (26). The timing and intensity of the response varies, depending on the activation route (22), but in both cases an increase occurs in the intracellular glycerol concentration that allows the cells to grow in high-osmolarity media. This response is mediated by an increase in the transcription of GPD1 (1), which encodes the enzymatic activity that controls glycerol production. In addition, the expression of several other genes dependent upon this pathway is also increased, among them CTT1 (35) and GLO1 (14). The major transcription factor(s) controlled by the HOG pathway has not yet been identified in S. cerevisiae, although there is clear evidence that suggests that part of the transcriptional induction of the GPD1 gene is the result of relief of the repression caused by the Tup1p-Ssn6p complex (24).

In addition to its role in osmoregulation, the HOG pathway has also been implicated in the architecture of the S. cerevisiae cell wall through its control over -glucan synthesis. PBS2 has been implicated in the transcriptional regulation of EXG1, a gene that encodes the major yeast glucanase. This regulation is translated into significant changes in (1-6)glucan contents, depending on the deletion or overexpression of the PBS2 gene (15). Similarly, deletion or overexpression of this gene leads to significant differences in (1-3)glucan synthase and (1-3)glucan levels through an unknown mechanism (16). This involvement of the HOG pathway in cell wall architecture together with the activation of the PKC pathway under hypo-osmotic conditions (10) suggests an interconnection between these two signaling pathways. Based on this and other evidence, a model has very recently been proposed in which the PKC and HOG pathways would have opposite roles in the balancing of cell wall plasticity (28).

The present work focuses on the characterization of the resistance to calcofluor of S. cerevisiae mutants affected in the HOG pathway and shows that the antifungal effect of calcofluor depends not only on chitin, as previously suspected, but also, at least partially, on the presence of this signal transduction pathway. In addition, we show that the HOG pathway maintains its functionality under noninducing conditions, guaranteeing cellular protection against sudden increases in osmolarity (osmotic tolerance) and contributing to the maintenance of the cell wall architecture.

	MATERIALS AND METHODS

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Yeast strains, media, and general methods. The yeast strains used in this work are described in Table 1. pbs2 null mutants in different genetic backgrounds were obtained by gene disruption as described elsewhere (35). Typical yeast media were used: yeast extract-peptone-dextrose (YEPD) as a complex medium and synthetic dextrose (SD) as a minimal medium, to which the corresponding supplements were added. Typically, experiments involving different NaCl concentrations were carried out on YEPD medium, while resistance to calcofluor was always measured on SD solid medium supplemented with 50 mM biphthalate buffer, pH 6.1, and different concentrations of calcofluor (31). Unless specified, all experiments were carried out with logarithmically growing cells (1 × 10⁷ to 2.5 × 10⁷/ml).

Plasmids. Plasmid pCTT1-18/7x (35) was integrated into the chromosome after linearization with NcoI. Plasmids pLJ1 (pRS316::PBS2) and pLJ2 (pRS426::PBS2) were obtained by direct cloning of PBS2 as a SpeI/SacI fragment obtained from plasmid L42 (16). pLJ3 (pRS316::PBS2-14) and pLJ4 (pRS426::PBS2-14) contain a mutated copy of PBS2 generated by site-directed mutagenesis. The cloning of PCR products was accomplished in the pGEM-T plasmid (Promega Corporation) following the manufacturer's instructions.

Assays for tolerance to osmotic dehydration. Tolerance to osmotic dehydration (osmotic tolerance) (3) is defined as the capacity of a culture to grow in dehydration medium (YEPD agar supplemented with 1.4 M NaCl) and is expressed as the percentage of cells that grow in this medium compared to that grown in YEPD agar. Alternatively, sorbitol-supplemented (YEPD agar-2.2 M sorbitol) plates were used as a dehydration medium. Typically, a culture growing logarithmically was diluted, and after one generation time, the culture was assayed for its osmotic tolerance after appropriate dilution by plating different aliquots onto YEPD or dehydration medium. Colonies were counted after 48 h (YEPD medium) or 7 days (dehydration medium) of growth. The osmotic tolerance of log-phase cells is defined as basal osmotic tolerance. When required, different aliquots of this log-phase culture were supplemented with the compound to be tested and osmotic tolerance was measured at different times (see Results for details of specific protocols). Osmotic tolerance in early-stationary-phase cells was assayed after 7 h of growth in YEPD medium. Cells containing plasmids were pregrown in selective medium, but dilution and further incubation were carried out in YEPD medium.

Analytical determinations. Chitin contents were measured as the amount of N-acetyl-glucosamine present in the alkali-insoluble cellular fraction after its complete digestion with chitinase. The whole protocol has been described previously (38).

Immunological procedures. Pbs2p and Pbs2-14p were detected in total cellular extracts as follows. Total-protein samples were obtained as described previously (9), quantified by absorbance at 280 nm, and loaded in equivalent amounts into sodium dodecyl sulfate-7.5% polyacrylamide gels. After electrophoresis, the proteins were transferred to an Immobilon-P membrane (Millipore), incubated with anti-Pbs2p goat polyclonal antibody (PBS2-yN-19; Santa Cruz Biotechnology), and immunodetected by the ECL method (Amersham Life Science).

Isolation of multicopy suppressors of the pbs2-14 mutation. Multicopy suppressors of the pbs2-14 mutation were isolated as follows. Strain EJY14 was transformed with an S. cerevisiae library constructed in the multicopy plasmid YEp24 (6). Transformants were grown on selective medium, and individual clones were screened for resistance to calcofluor. The clones that were sensitive to 0.5 mg of calcofluor/ml were subjected to plasmid loss by growing them on 5-fluoro-orotic acid plates. All the clones in which sensitivity to calcofluor was associated with the presence of a plasmid were selected for further study. We isolated five clones that might contain bona fide suppressors. In these cases, the plasmids were isolated and both ends of the cloned regions were sequenced using primers that anneal onto vector sequences. Comparison of the region sequenced with databases allowed us to pinpoint the exact restriction map of the clones, and a specific subcloning strategy was therefore designed for each suppressor. The open reading frame contained in the minimum DNA fragment able to complement the calcofluor resistance of strain EJY14 was considered to be the suppressor.

	RESULTS

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Isolation of mutants resistant to calcofluor. The strategy of isolating mutants resistant to calcofluor has been very useful in S. cerevisiae for the identification of genes involved in chitin biosynthesis (8). However, this strategy has not been fully exploited because it is time-consuming in the sense that more than 90% of mutants arise because of mutations in the CHS3 gene (38). To avoid this problem, we designed a new screening procedure using a strain, LJY3, that contains two copies of CHS3: the chromosomal copy and another carried by pHV7 (39). In addition, selection was carried out in synthetic complete medium supplemented with only 0.5 mg of calcofluor/ml. Using this strategy, we isolated 63 new mutants resistant to calcofluor that, after rechecking, were back-crossed with all previously known chs mutants. Analysis of the diploids formed indicated that 48 of the mutants were affected in previously defined loci (38), and their study was therefore abandoned (Table 2). The 15 remaining mutants could be classified in two groups in terms of septum enlargement after calcofluor treatment (Table 2, last column). Previous studies had indicated that the absence of septum enlargement is usually associated with a defect in chitin synthesis (9, 32). This work focuses on the group of calcofluor-resistant mutants without apparent defects in chitin synthesis.

HOG pathway mutants show different degrees of resistance to calcofluor but do not have alterations in chitin synthesis. In order to understand the mechanism(s) involved in the resistance to calcofluor observed in the above-mentioned mutants, we tested the resistance levels of several HOG pathway mutants (Fig. 1). Clearly, the pbs2 null mutant can be classified as calcofluor-resistant, since it was able to grow at 0.5 mg of Calcofluor/ml (Fig. 1) and even up to 0.75 mg/ml (data not shown). However, growth at 1.0 mg/ml, a concentration at which the chs3 mutant can grow, was severely impaired (data not shown). sho1 and ssk2 ssk22 mutants also showed partial resistance to calcofluor, and this resistance increased significantly in the triple sho1 ssk2 ssk22 mutant. Unfortunately, we were unable to compare the absolute resistance values due to significant differences in the genetic backgrounds of the strains (Fig. 1, compare the respective wild types, W303 and TM141). By contrast, mutants with mutations in the GPD1 gene, a known target of the HOG pathway, were calcofluor sensitive (Fig. 1), as were the gpd2 and gpd1 gpd2 null mutants (data not shown).

View larger version (74K): [in this window] [in a new window]   FIG. 1.   S. cerevisiae growth on plates supplemented with different calcofluor concentrations. Serial dilutions of each strain as indicated above the panels were plated onto complete SD medium supplemented with different calcofluor concentrations as indicated below the panels, incubated for 48 h at 28°C, and photographed. Note the different genetic backgrounds used: W303 (upper) and TM141 (lower). wt, wild type.

Calcofluor induces osmotic tolerance and increased glycerol concentrations but does not induce HOG pathway activation. Activation of the HOG pathway by increased external osmolarity induces two clear physiological effects, an increase in the concentration of intracellular glycerol (1) and an increase in osmotic tolerance (20). To test the effect of calcofluor on osmotic tolerance, we incubated early logarithmically growing cells in YEPD supplemented with 0.075 mg of calcofluor/ml and determined their osmotic tolerances (see Materials and Methods) after different treatment times. Calcofluor treatment induced osmotic tolerance in the wild-type strain in a time-dependent fashion, since the basal levels of osmotic tolerance (zero time) were increased by approximately 30-fold after 105 min of treatment (Fig. 2 and Table 4). A similar increase in osmotic tolerance was obtained when plates supplemented with sorbitol were used (data not shown). The increase in osmotic tolerance due to calcofluor treatment was not observed in the chs3 mutant, although the basal levels in this strain were considerably higher (Fig. 2A and Table 4). This increase was very significant, although it was considerably less than that observed after induction of the HOG pathway by NaCl treatment (20) (Fig. 2B and Table 4). Apparently, the binding of calcofluor to chitin is required for the observed increase in osmotic tolerance.

View larger version (16K): [in this window] [in a new window]   FIG. 2.   Osmotic tolerance of different S. cerevisiae strains. (A) Osmotic tolerance of a logarithmically growing culture after incubation in the presence of 0.075 mg of calcofluor/ml for the indicated times. (B) Osmotic tolerance after osmotic (0.4 M NaCl) treatment for the indicated times. Osmotic tolerance is expressed as the percentage of cells able to grow on dehydration medium compared to those growing on YEPD. See Materials and Methods for details. , W303 (wild type); , TC1 (chs3; , LJYE14 (pbs2-14).

Characterization of the pbs2-14 mutant. We have shown above that strain E-14 contains a mutation that confers resistance to calcofluor but also supports growth at high salt concentrations (Fig. 1 and Table 2) or at 1.5 M sorbitol (data not shown). Diploid strain E14/LJY1 (pbs2::LEU2) is resistant to calcofluor, as are the parent strains, indicating that E14 contains a mutation in the PBS2 gene. To confirm this, segregation analysis was carried out on this diploid, resulting in a calcofluor resistance segregation of 4:0. In addition, the resistance of strain E14 to calcofluor was reversed after transformation with a centromeric plasmid containing the PBS2 gene. Therefore, strain E14 must contain a new pbs2 mutant allele; we call it pbs2-14.

View larger version (41K): [in this window] [in a new window]   FIG. 3.   Molecular characterization of the PBS2-14 mutation. (A) Schematic representation of PBS2-14 PCR cloning. Primer pairs and the conserved protein kinase (PK) domain are indicated. The arrow shows the approximate position of the E14 mutation. (B) Sequence discrepancy between wild type and mutant; the deleted base is indicated by an arrow. The protein translation is shown on the right side of each picture. (C) C-terminal sequence of Pbs2 and Pbs2-14 proteins. (D) Immunodetection of Pbs2p and Pbs2-14p in Western blot using anti-Pbs2p antibodies (see Materials and Methods). The arrowheads indicate the Pbs2 protein that is absent in the pbs2 null mutant. The altered Pbs2-14 protein is indicated by an asterisk. WT, wild type.

View larger version (37K): [in this window] [in a new window]   FIG. 4.   Mutant growth in different media after transformation with different plasmids. Serial dilutions of each strain were plated onto SD medium supplemented with 0.5 mg of calcofluor/ml or YEPD medium supplemented with 0.9 M NaCl. The plates were photographed after 48 h of growth at 28°C. The strains are indicated above each picture. The plasmids carried by each strain are indicated at the right. (A) Calcofluor and salt plates are shown (W303 background). (B) Only calcofluor plates are shown. Note that TM285, TM257, and TM310 strains are in the TM141 background.

Isolation of multicopy suppressors of the pbs2-14 mutation. In an attempt to identify some of the genes involved in calcofluor sensitivity and resistance through the HOG pathway, we searched for multicopy suppressors of the pbs2-14 mutation. To do so, we transformed strain LJYE14 with an S. cerevisiae gene bank constructed in the episomic vector YEp24 and looked for transformants sensitive to calcofluor. We selected several transformants that were more sensitive to calcofluor than the parent strain (Fig. 5). After standard procedures (plasmid loss, end sequencing, and subcloning), we were able to assign the specific suppressors to four independently isolated genes, ECM30, CDC11, MSS4, and RED1, none of which were previously associated with the HOG pathway. The ECM30 gene has been identified in a screening for insertion mutants hypersensitive to calcofluor (19). CDC11 is a member of the septin family and hence is involved in the organization of the septum during cell division (18). MSS4 encodes a phosphatidylinositol-4-phosphate 5-kinase that is required for cell morphogenesis (12). Finally, RED1 encodes a protein required for chromosome segregation during meiosis, although its exact function is unknown (37). The four genes clearly suppressed the calcofluor resistance phenotype associated with the pbs2-14 mutation but also weakly suppressed the resistance observed in the null pbs2 mutant (Fig. 5). In addition, these multicopy suppressors also conferred weak hypersensitivity to calcofluor when introduced in wild-type strains (data not shown).

View larger version (54K): [in this window] [in a new window]   FIG. 5.   Effects of different multicopy suppressors on calcofluor resistance. Serial dilutions of each strain were plated onto SD selective medium supplemented with different calcofluor concentrations (shown at bottom). The recipient strains (top) were transformed with multicopy plasmids containing the genes shown on the left.

View larger version (63K): [in this window] [in a new window]   FIG. 6.   Epistatic relationship between the PKC and HOG signal transduction pathways. (A) Serial dilutions of each strain (15Dau background) were plated onto YEPD medium, and replica plates were incubated at the indicated temperatures for 48 h. (B) Isogenic W3031A or LJY1 (pbs2::LEU2) strains were transformed with plasmid pNV7-MKK1P386 in which the hyperactive form of the MKK1 gene is under the control of the GAL1 promoter (40). Serial dilutions of each strain were plated onto selective medium with either glucose or galactose as a carbon source. Growth was scored after 2 (glucose) or 4 (galactose) days of growth.

	DISCUSSION

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The HOG pathway is involved in S. cerevisiae resistance and sensitivity to calcofluor. Previous attempts to isolate S. cerevisiae mutants resistant to calcofluor have been successful in the identification of genes involved in chitin synthesis (32, 38). The fact that all the complementation groups identified are deficient in chitin synthesis prompted us to assume that chitin is the primary and only target of this drug (8). However, in addition to the chitin defect, it should be possible to obtain resistance by blocking the increase in chitin synthesis induced by calcofluor treatment. This hypothesis is sustained by earlier reports that indicate that calcofluor binds chitin, increases its synthesis, and disturbs the cell wall (30, 31). Very recently, new mutants partially resistant to calcofluor but with no apparent defect in chitin synthesis have been isolated (19). However, this study focused on the isolation strategy and not on mutant characterization, and it would therefore be unwise to attempt to draw any conclusions about the mechanism(s) of action of calcofluor based on these data alone.

Physiological role of the HOG pathway under noninducing conditions. Sequencing of the pbs2-14 mutation indicated that Pbs2-14p is only altered in its C-terminal domain; therefore, the mutation should not affect the catalytic domain of the kinase (4). This is in agreement with the growth of the pbs2-14 mutant at high salt concentrations. Figure 2 shows that osmoadaptation after salt treatment is normal in this mutant and follows kinetics similar to those of the wild-type strain. In addition, induction of the STRE-LacZ fusion and the increase in intracellular glycerol levels by salt in this mutant are comparable to those observed in the wild-type strain (data not shown). All these observations are clear indications that activation of the HOG pathway by increased osmolarity occurs normally in the pbs2-14 mutant.