J. C. Kapteyn , , E-mail firstname.lastname@example.org; Tel. (+31) 20 525 7850; Fax (+31) 20 525 7934.
In Candida albicans wild-type cells, the β1,6-glucanase-extractable glycosylphosphatidylinositol (GPI)-dependent cell wall proteins (CWPs) account for about 88% of all covalently linked CWPs. Approximately 90% of these GPI-CWPs, including Als1p and Als3p, are attached via β1,6-glucan to β1,3-glucan. The remaining GPI-CWPs are linked through β1,6-glucan to chitin. The β1,6-glucanase-resistant protein fraction is small and consists of Pir-related CWPs, which are attached to β1,3-glucan through an alkali-labile linkage. Immunogold labelling and Western analysis, using an antiserum directed against Saccharomyces cerevisiae Pir2p/Hsp150, point to the localization of at least two differentially expressed Pir2 homologues in the cell wall of C. albicans. In mnn9Δ and pmt1Δ mutant strains, which are defective in N- and O-glycosylation of proteins respectively, we observed enhanced chitin levels together with an increased coupling of GPI-CWPs through β1,6-glucan to chitin. In these cells, the level of Pir-CWPs was slightly upregulated. A slightly increased incorporation of Pir proteins was also observed in a β1,6-glucan-deficient hemizygous kre6Δ mutant. Taken together, these observations show that C. albicans follows the same basic rules as S. cerevisiae in constructing a cell wall and indicate that a cell wall salvage mechanism is activated when Candida cells are confronted with cell wall weakening.
The cell wall of the opportunistic human pathogenic fungus Candida albicans is composed of complex polymers of glucose (β1,3- and β1,6-glucan), chains of N-acetylglucosamine (chitin) and cell wall mannoproteins (Shepherd, 1987; Fleet, 1991). The β-glucans are the main constituents, accounting for 50–60% by weight of the cell wall. Together with chitin, a relatively minor component (0.6–3%), the glucans form a rigid skeleton, responsible for the shape of the cell and its physical strength (Shepherd, 1987). The mannoproteins represent 30–40% of the wall and determine the cell surface properties, enabling Candida albicans cells to adhere to host tissues and to immunomodulate the host immune response (Chaffin et al., 1998). Our knowledge about the molecular organization of the Candida cell wall is limited, but considerable progress has been made in unravelling the cell wall architecture of Saccharomyces cerevisiae (Klis et al., 1997; Kollár et al., 1997; Lipke and Ovalle, 1998; Kapteyn et al., 1999a,b; Smitset al., 1999). S. cerevisiae is generally considered to be a valuable model organism for studying cell wall construction in Candida (Klis et al., 1997; Chaffin et al., 1998). Recently, a tentative model of the molecular organization of the S. cerevisiae cell wall has been presented (Fig. 1; Lipke and Ovalle, 1998; Smits et al., 1999). According to this cell wall model, the β1,3-glucan molecules form a three-dimensional matrix surrounding the entire cell. As these β1,3-glucan molecules are branched, they may have multiple non-reducing ends. These may function as attachment sites for cell wall proteins (CWPs). Two main types of covalently linked CWPs have been identified, namely glycosylphosphatidylinositol (GPI)-dependent CWPs and Pir-CWPs (Kapteyn et al., 1999b). The GPI-CWPs are attached via β1,6-glucan to a non-reducing end of a β1,3-glucan molecule (Fig. 1; Kapteyn et al., 1996, 1999b; Kollár et al., 1997). A GPI-derived structure is responsible for the linkage of this type of proteins to β1,6-glucan (Kollár et al., 1997; Fujii et al., 1999). The Pir-related CWPs are directly attached to β1,3-glucan through an, as yet unknown, base-labile linkage (Fig. 1; Mrsa et al., 1997; Kapteyn et al., 1999a). After cytokinesis, chitin molecules may also attach to the non-reducing ends of the β1,3-glucan molecules and, less frequently, also to the non-reducing ends of highly branched β1,6-glucan molecules (Kollár et al., 1995, 1997), thereby strengthening the cell wall (Fig. 1; Lipke and Ovalle, 1998; Smits et al., 1999).
The cell wall structure of S. cerevisiae is highly flexible. For instance, yeast cells have been shown to be able to compensate for defects in β1,3-glucan synthesis and/or assembly by altering the molecular organization of the wall. This compensation is characterized by a several-fold increase in chitin content (Popolo et al., 1997) and in an augmented number of linkages between chitin and β1,6-glucan, the attachment moiety for GPI-CWPs (Kapteyn et al., 1997; Lipke and Ovalle, 1998; Ram et al., 1998). As a consequence of this compensatory reaction, a 20-fold increase was observed in the level of GPI-CWPs that were resistant to β1,3-glucanase-extraction (Kapteyn et al., 1997). When yeast cells were confronted with loss or faulty assembly of β1,6-glucan, resulting in the release of GPI-CWPs into the medium, a strong increase in the level of β1,3-glucan-bound Pir proteins was observed (Kapteyn et al., 1999a). In addition, chitin deposition was also massively enhanced (Kapteyn et al., 1999a; Ketela et al., 1999). This latter phenotype is shared by many other yeast cell wall-defective mutants (Ram et al., 1994; Gentzsch and Tanner, 1996; Lussier et al., 1997; Dallies et al., 1998). The observed increase in chitin levels may be mediated by stimulation of Chs3p activity through the putative cell wall stress sensor Mid2p (Ketela et al., 1999). High chitin levels have also been reported for a β1,6-glucan-deficient Candida glabrata kre9Δ strain (Nagahashi et al., 1998) and a C. albicans phr1Δ mutant, which is defective in β-glucan assembly (Popolo and Vai, 1998).
In this paper, we present evidence for C. albicans following the same rules as S. cerevisiae in constructing a cell wall. In Candida cells, ≈ 88% of all covalently linked CWPs, including proteins encoded by the ALS gene family, are immobilized in the wall as GPI modules, whereas the remaining proteins are attached as Pir modules. At least two CWPs were identified, showing significant homology to S. cerevisiae Pir2p/Hsp150. In cell wall-defective mutants, we observed changes in the relative frequency of GPI and Pir proteins, enhanced chitin levels and increased cross-linking of GPI-CWPs through β1,6-glucan to chitin. These and other data therefore suggest that, like in S. cerevisiae, mechanisms are activated by which C. albicans is able to counteract cell wall damage.
Identification of putative GPI-dependent CWPs, including Als1p and Als3p, in cell walls of C. albicans
Fig. 1 shows the current model of the S. cerevisiae cell wall. Our experiments were aimed at determining whether the same model applies to C. albicans. In order to demonstrate that C. albicans contains β1,6-glucan-linked GPI-dependent CWPs, we analysed β1,6-glucanase digests of SDS-extracted cell walls from C. albicans CAI4 yeast forms grown in SC medium at 28°C. Western blotting using Concanavalin A (ConA) peroxidase revealed a large smear and four relatively discrete mannosylated protein bands with an apparent molecular mass of 220, 170, 100 and 65 kDa respectively (Fig. 2A, lane 1). Anti-β1,6-glucan antiserum did not recognize any of this material, indicating that the β1,6-glucanase did not leave any detectable β1,6-glucan epitope attached to the proteins (data not shown).
Hoyer et al. (1998a,b, 1999) have shown the existence of a family of cell surface proteins that are probably involved in cell adhesion. In view of their homology to α-agglutinin, a sexual adhesion protein, the corresponding genes have been called agglutinin-like sequences (ALS genes). Reprobing of the stripped blot with an anti-Alsp antiserum (Hoyer et al., 1998b) revealed a band at ≈ 600 kDa (Fig. 2B, lane 1). Endoglycosidase (Endo) H treatment of the Als protein band reduced its apparent molecular mass by ≈ 120 kDa and demonstrated the presence of N-linked carbohydrate (Fig. 2B, lane 2). Because the anti-Als antiserum recognizes various Als proteins (Hoyer et al., 1999), Northern blot analysis was performed to determine which ALS genes were transcribed in SC-grown yeast forms. In a first screen, we used the ALS1 tandem repeat probe and the ALS5 tandem repeat probe, each one defining a separate ALS subfamily (Hoyer et al., 1998b). Signals were only observed on the blot hybridized with the ALS1 repeat fragment. The sizes of the bands were consistent with the ALS1-specific messages produced by the two alleles of ALS1 in strain CAI4 (Hoyer et al., 1998a). The expression of ALS1 was confirmed by incubating the blot with an ALS1-specific probe (data not shown). These data support the conclusion that the protein detected in the Western blot is Als1p. In order to demonstrate that also O-glycosylated side chains contribute to the molecular mass of mature Als1p, we analysed Als1p from a C. albicans pmt1Δ mutant, which is perturbed in O-glycosylation of proteins (Timpel et al., 1998). Western blot analysis revealed that Als1p released by β1,6-glucanase from this mutant strain was more heterogeneous and about 60 kDa smaller in size than Als1p from the parental strain (Fig. 2B, compare lanes 3 and 4), which is consistent with the idea that the protein is O-glycosylated. The response of the pmt1Δ mutant is not caused by the expression of additional Als proteins as we did not see any difference in the transcription of ALS genes in the wild type or in mutant strains (data not shown).
Similar analyses were conducted on C. albicans CAI4 cells grown for 4 h at 37°C in RPMI medium, allowing the additional expression of ALS3 (Hoyer et al., 1998a). At our inoculation density of 108 cells ml−1, C. albicans grew mainly as a yeast form, but with germ tube and hyphal forms commonly present in the culture. β1,6-Glucanase extracts of RPMI-grown cells yielded a slightly different CWP profile when reacted with ConA peroxidase (Fig. 2A, compare lanes 1 and 2). For example, the high molecular weight material in the upper part of the gel migrated further, whereas around 100 and 65 kDa differences in staining intensity were observed (Fig. 2A). Treatment of the stripped blot with anti-Als serum showed two immunoreactive bands with molecular masses of about 600 and 440 kDa respectively (Fig. 2B, lane 5). To further identify these putative Als proteins, we performed a two-step Northern analysis. Using the subfamily-specific ALS1 and ALS5 tandem repeat probes in the first round and the gene-specific probes for ALS1 and ALS3 in the second round, we identified two different transcripts originating from ALS1 and ALS3 respectively (data not shown). These data are consistent with previous gene expression data (Hoyer et al., 1995, 1998a). As the ALS3 sequence is shorter than that of ALS1, these data indicate that the larger protein in each blot is Als1p, whereas the smaller protein in the RPMI blot is Als3p. Because both proteins are extractable from the cell wall with β1,6-glucanase digestion, these Als proteins fit the model of immobilization in the cell wall as GPI modules.
Identification of Pir-related CWPs in C. albicans
Because S. cerevisiae contains several β1,3-glucan-bound Pir-related CWPs (Mrsa et al., 1997; Kapteyn et al., 1999a), we looked for similar proteins in C. albicans. For these studies, we utilized the β1,6-glucanase-resistant β1,3-glucanase-extractable CWPs from both RPMI- and SC-grown cultures and an anti-Pir2p antiserum, which is known to recognize Pir2p-related proteins in various yeast species (Russo et al., 1992). Western blots of proteins from the RPMI culture yielded a single band with an apparent molecular weight of 235 kDa (Fig. 3, lane 1). After Endo H treatment, the molecular mass of the protein decreased to about 200 kDa (Fig. 3, lane 2). Before and after Endo H treatment, the antigenic Pir2p homologue detected was also reactive with an antiserum raised against β1,3-glucan, indicating that the protein was associated with a β1,3-glucan moiety (Fig. 3, lanes 6 and 7). However, no immunoreactive bands were observed with the anti-β1,6-glucan antiserum (data not shown). Upon very mild alkali treatment, the β1,3-glucan epitope was lost (Fig. 3, lane 8), which was accompanied by a drop in molecular size of the Endo H-treated Pir2 homologue by ≈ 50 kDa (Fig. 3, lane 3). These data strongly suggest that C. albicans incorporates at least one Pir-related protein into its cell wall through an alkali-sensitive linkage to β1,3-glucan. Clear labelling of the C. albicans cell wall by immunogold electron microscopy with the anti-Pir2p serum supports this conclusion (Fig. 4A and B). The intensity of the immunogold labelling is, however, slightly less than that observed in cell walls of S. cerevisiae (Fig. 4C and D). Hardly any gold labelling was observed with preimmune serum (data not shown). These findings are therefore in accordance with earlier data from Mormeneo et al. (1994), who were the first investigators to release proteinaceous material from Candida by mild alkali extraction.
Interestingly, when cells were grown in SC medium, pH 4.5, two immunoreactive bands were observed in the β1,6-glucanase-resistant β1,3-glucanase-extractable wall fraction, which upon Endo H treatment had apparent molecular weights of 200 and 320 kDa, respectively (Fig. 3, lanes 4 and 5), suggesting the existence of more than one Pir protein in C. albicans. Further research is required to establish whether C. albicans indeed contains a family of PIR genes as has been described for S. cerevisiae (Toh et al., 1993; Mrsa et al., 1997). Notably, homology searches of the S. cerevisiae PIR1, PIR2, PIR3 and PIR4/CCW5/CCW11 genes with sequences of the Candida genome data bank (http://candida.stanford.edu/ybc.html) resulted in the identification of a putative C. albicans PIR-related ORF, 265178D12.yl.seq (data not shown). The translated sequence of this ORF has 32–45% identity to, and 48–60% similarity with, the four Pir-CWPs of S. cerevisiae. The C. albicans PIR-related ORF is predicted to encode a secretory polypeptide of 104 amino acids with a putative N-terminal signal sequence of 18 amino acids (psortii).
Changes in cell wall architecture of cell wall-defective mutants
Because the cell wall of S. cerevisiae is a highly dynamic structure, we reasoned that the C. albicans cell wall might also have a flexible architecture. To explore this possibility, we utilized several C. albicans mutant strains, including mnn9Δ (defective in N-glycosylation of proteins), pmt1Δ (defective in O-glycosylation) and a hemizygous kre6Δ strain, which has an 80% reduction in the level of β1,6-glucan (Mio et al., 1997). As expected from earlier reports on S. cerevisiaeβ1,6-glucan-deficient kreΔ mutants (Lu et al., 1995; Jiang et al., 1996; Kapteyn et al., 1999a), the hemizygous kre6Δ mutant secreted large amounts of Als proteins into the culture supernatant (Fig. 5A). Because these cells were grown in RPMI medium, the secreted immunoreactive proteins probably represent both Als1p and Als3p. These observations provide additional evidence that Als proteins are immobilized in the cell wall through linkage to β1,6-glucan. No enhanced secretion of Als proteins was observed for the pmt1 or mnn9 null strains (data not shown).
To investigate the molecular cell wall organization of C. albicans CAI4 and the selected mutants, we first measured chitin levels in SC-grown cells. The glycosylation-deficient pmt1 and mnn9 null mutants showed a two- to threefold increase in the level of chitin. Consistent with an essential role of chitin in strengthening the wall, the pmt1 and mnn9 null strains were found to be more sensitive to exochitinase and nikkomycin Z, a competitive inhibitor of chitin synthases (Choi et al., 1994; Gaughran et al., 1994), than the parental strain (data not shown). The chitin level in the hemizygous kre6Δ strain was unaffected (Table 1) and consistent with this the hemizygous kre6Δ strain was as sensitive to nikkomycin Z as the parental strain (data not shown). Lack of altered chitin levels in the hemizygous kre6Δ strain was unexpected because similar mutations in S. cerevisiae haploid strains result in increased chitin content (Dallies et al., 1998; Kapteyn et al., 1999a; Ketela et al., 1999). Next, we investigated whether the increased chitin levels in pmt1 and mnn9 null mutants were accompanied by an increase in the amount of β1,3-glucanase-resistant GPI-CWPs because of an enhanced coupling of these proteins through β1,6-glucan to chitin. In wild-type cells and in the hemizygous kre6Δ strain, about 8–9% of the total radiolabelled CWPs were resistant to β1,3-glucanase extraction, whereas this percentage was increased to 21% and 33% in the pmt1 and the mnn9 null mutants respectively (Table 1). Subsequent treatment with exochitinase resulted in the solubilization of almost all β1,3-glucanase-resistant proteins (Table 1). Consistent with these results, Western analyses demonstrated that the β1,3-glucanase-resistant exochitinase-soluble mnn9Δ (data not shown) and pmt1Δ wall fractions contained higher levels of β1,6-glucosylated ConA-reactive GPI-CWPs (Fig. 6A and B), including Als1p (Fig. 6C). Notably, the ConA-reactive CWP bands released from the pmt1Δ strain were generally smaller and more heterogeneous than those derived from the parental strain (Fig. 6A).
Table 1. . Chitin content and extractability of SDS-resistant 14C-labelled CWPs by sequential cell wall digestions with different hydrolytic enzymes (comparison of Candida albicans cells and cell wall-defective mutants grown in SC medium at 28°C).
We also determined the level of β1,6-glucanase-resistant β1,3-glucanase-extractable Pir proteins in the strain CAI4 and in the selected C. albicans mutants. In CAI4, about 12% of the CWPs were resistant to β1,6-glucanase digestion, a value that is slightly lower than that observed in S. cerevisiae wild-type cells (Kapteyn et al., 1999a). Similar amounts of β1,6-glucanase-resistant CWPs were only slightly enhanced in the mutant strains (Table 1). This result was confirmed using Western analyses, which showed that the β1,6-glucanase-resistant β1,3-glucanase extract from both the glycosylation mutants (data not shown) and the hemizygous kre6Δ strain contained moderately increased levels of the putative Pir2 homologue compared with the corresponding wild-type fraction (Fig. 5B).
In this study, we investigated whether the structure of the C. albicans cell wall resembles the molecular organization of the S. cerevisiae cell wall (Fig. 1; Lipke and Ovalle, 1998; Smits et al., 1999). We found many similarities between the two cell wall structures. As in S. cerevisiae, the C. albicans cell wall contains GPI-CWP modules that are linked to β1,6-glucan (Fig. 2). C. albicans also contains Pir-CWP modules that are linked through an alkali-labile linkage to β1,3-glucan and are resistant to β1,6-glucanase treatment (Figs 3 and 4). We demonstrated immunologically the existence of at least two Pir-CWPs. It was also found that, like S. cerevisiae, C. albicans is able to alter its cell wall structure in response to cell wall damage or weakening (Table 1; Figs 5 and 6). A significant difference between the two cell wall structures, however, is that the relative amount of β1,3-glucanase-resistant CWPs in C. albicans is approximately fourfold higher than that in S. cerevisiae wild-type cells (Table 1; Kapteyn et al., 1997, 1999a). As both organisms have similar chitin levels, this suggests that there are more linkages between chitin and β1,6-glucan in C. albicans than in S. cerevisiae. The two- to threefold increased chitin deposition in cell walls of the glycosylation-deficient C. albicans strains led to a proportional increase in the amount of β1,3-glucanase-resistant CWPs, suggesting that the additional chitin molecules synthesized under these cell wall stress conditions are coupled to β1,6-glucan rather than to β1,3-glucan.
As well as defining the basic architecture of the C. albicans cell wall, our studies also contributed to a greater understanding of the structure and localization of Als proteins. The work presented here leads to the following conclusions about these proteins: they are linked to β1,6-glucan and they are N- and O-glycosylated. Amino acid sequences predicted from the ALS genes are consistent with these conclusions. Each Als protein encodes a hydrophobic C terminus with characteristics of a GPI-anchor-addition site (Hoyer et al., 1998b). Although our data do not precisely identify an Als protein intermediate with an attached GPI moiety, they serve to emphasize the parallels between what is known for S. cerevisiae GPI-CWPs and the C. albicans Als family proteins. Als proteins are linked in the C. albicans cell wall in a manner consistent with proteins that carry a transiently linked GPI moiety. N-Glycosylation of Als proteins is probable due to the presence of consensus N-glycosylation sites, predominantly in the tandem repeat and C-terminal domain regions of each protein (Hoyer et al., 1995, 1998a,b). O-Glycosylation of Als proteins is assumed because of the serine/threonine-rich content of the tandem repeat and C-terminal domains (Hoyer et al., 1995, 1998a,b).
Although a clear picture is beginning to develop as to how CWPs are immobilized in the cell wall of C. albicans and S. cerevisiae, the function of most of these proteins is unknown (Chaffin et al., 1998; Kapteyn et al., 1999b; Smits et al., 1999). Proteins of the C. albicans Als family may constitute an exception, however, as heterologous expression of ALA1/ALS5 and ALS1 in S. cerevisiae confers adherence to human cells (Gaur and Klotz, 1997; Fu et al., 1998), suggesting that ALS genes may encode adhesins. Recently, Hwp1p, another GPI-dependent CWP of candidal germ tubes and hyphae, was demonstrated to mediate covalent attachment of Candida cells to human epithelial tissue, as a substrate for human transglutaminase activity (Staab et al., 1999). In yeast, Pir-CWPs confer resistance to the plant antifungal protein osmotin (Yun et al., 1997). Sequential disruption of the PIR-related genes CCW5, PIR2/CCW7, PIR1/CCW6 and PIR3/CCW8 led to reduction in growth, swollen and irregular cell morphology and an increased susceptibility to calcofluor white (Mrsa and Tanner, 1999). In addition, the triple and quadruple deletants showed a diminished mating ability (Mrsa and Tanner, 1999). Expression of the best-studied Pir protein Pir2p is induced by a variety of stress conditions, including heat shock, nitrogen starvation, oxidative stress, aluminium and cell wall damage owing to reduced levels of β1,6-glucan (Russo et al., 1992, 1993; Ezaki et al., 1998; Kapteyn et al., 1999a). However, a clear function has been ascribed neither to Pir2p/Hsp150 nor to any of the other Pir proteins. Like S. cerevisiae Pir2p/Hsp150, the Candida Pir2-related protein seems to be expressed at a higher level under conditions of cell wall weakening (Fig. 5). Whether the expression of C. albicans Pir proteins is differentially regulated and whether these proteins play a role in the pathogenicity of C. albicans is currently being explored.
C. albicans strains and growth conditions
The Candida strains used are CAI4 (ura3Δ::imm434/ura3Δ::imm434), CAP1-312 (pmt1Δ::hisG-URA3-hisG/pmt1Δ::hisG in CAI4), SSCA-2 (mnn9Δ::hisG-URA3-hisG/mnn9Δ::hisG in CAI4) and 211 (KRE6/kre6Δ::hisG-URA3-hisG in CAI4). Cells were grown at 28°C in synthetic complete (SC) medium, containing 0.17% (w/v) yeast nitrogen base without amino acids and ammonium sulphate (Difco), 2% (w/v) glucose, 0.5% (w/v) casein hydrolysate, 0.5% (w/v) ammonium sulphate, 0.25% (w/v) succinate and 0.01% (w/v) uridine at pH 4.5. In some experiments, cells were precultured overnight in SC medium at 28°C, washed, resuspended and grown for a further 4 h at 37°C in RPMI-1640 medium (pH 7.2), which contains 2% (w/v) glucose, various inorganic salts and amino acids, as defined by the manufacturer (Gibco BRL), with an initial cell density of 108 cells ml−1.
Immunogold labelling of Pir2-related proteins
Cells grown in SC medium were taken up by capillary action in single dry cellulose capillary tubes of about 5–10 mm in length (Hohenberg et al., 1994). After filling, the capillary tube was submerged in 1-hexadecane and cut into pieces about 2.5 mm long. The 2.5 mm tubes with cells were sealed at their ends by pressing with a blunt razor blade. Four tubes were sandwiched in aluminium specimen holders. Thereafter, this sandwich was inserted in the holder of a Leica high-pressure freezer. After high-pressure freezing with a Leica EM HPF, the sandwich was put into liquid nitrogen (LN2). The two specimen holders were separated under LN2. The capillary tubes still attached to one of the specimen aluminium holders were freed from adhering 1-hexadecane under LN2 by gently scraping with a fine needle. Subsequently, the capillary tubes attached to the specimen holder were transferred to a screw-capped vial, which contained frozen substitution medium consisting of 0.3% uranyl acetate and 0.01% glutaraldehyde in anhydrous methanol and a miniature transfer basket (Hohenberg et al., 1994). This vial was placed in the substitution apparatus CS-auto (Sitte et al., 1985) at −90°C for 38 h. Subsequently, the temperature was raised to −35°C at a speed of 7°C h−1. The freeze-substitution medium was exchanged for methanol. The specimen holders were removed from the vial. The capillary tubes were infiltrated with an increasing concentration of Lowicryl HM20 (Carlemalm et al., 1982): 25% for 1 h; 50% for 1 h; 70% for 1 h and overnight; then at −20°C 100% for 2× 2 h; 100% over the weekend; freshly made 100% for 2 h. Thereafter, the capillary tubes were embedded in 100% Lowicryl HM20. Polymerization was carried out at −20°C for 42 h with a UV source attachment (Sitte et al., 1985). After 1 day of curing under UV light at room temperature, 80 nm sections were cut with a Diatome diamond knife and mounted on 1.1% pioloform-coated (Pioloform resin 2295/0; Polaron), carbon-evaporated 150 mesh nickel grids. After embedment, labelling was performed on sections of cells to locate Pir2-related proteins, using rabbit serum 1660 recognizing Pir2p/Hsp150 (1:500) (Russo et al., 1992). The antigen-antibody complexes were visualized with goat anti-rabbit antibodies (1 : 20) conjugated with 10-nm gold particles (Aurion).
Nucleic acid blots
Total RNA extraction, formaldehyde gel electrophoresis and Northern blotting were performed to analyse ALS gene expression (Hoyer et al., 1998a,1998b). Northern blots were first probed with an 875 bp KpnI fragment of pLH21 that encodes only ALS1 tandem repeat sequences (Hoyer et al., 1995) and a PCR-amplified fragment that includes sequences from the ALS5 tandem repeat region (Hoyer et al., 1998b). These probes each specifically recognize one of the two subfamilies of the ALS gene family: the ALS1 repeat probe fragment recognizes ALS1, ALS2, ALS3 and ALS4, whereas the ALS5 repeat probe fragment recognizes ALS5, ALS6 and ALS7 (Hoyer et al., 1998b; L. L. Hoyer, unpublished). Based on the signals observed with the initial screening with the tandem repeat probes, positive signals were precisely identified using ALS gene-specific probes. In this case, an ALS1-specific XbaI–HindII fragment from the 3′ end of ALS1 (Hoyer et al., 1995) and a PCR-amplified ALS3-specific fragment from the 3′ end of the coding region (Hoyer et al., 1998a) were used. This procedure allows the correct identification of the seven known ALS genes.
Isolation of cell wall proteins
Cells were harvested, washed and homogenized as has been described before (Montijn et al., 1994; Kapteyn et al., 1995). Subsequently, cell walls were isolated and washed extensively with 1 M NaCl. Isolated cell walls were boiled twice in the presence of SDS, EDTA and β-mercaptoethanol to solubilize the non-covalently linked CWPs and to remove any contaminants derived from the cytosol and/or plasma membrane (Mrsa et al., 1997; Klis et al., 1998). SDS-extracted cell walls were washed and treated with recombinant Trichoderma harzianum endo-β1,6-glucanase [0.8 U g−1 (wet weight of cell walls)], and purified according to the procedure described by Bom et al. (1998) to isolate the GPI-CWPs (Kapteyn et al., 1996). Subsequently, β1,6-glucanase-treated cell walls were digested with Quantazyme (Quantum Biotechnologies) [1500 U g−1 (wet weight of cell walls)], a recombinant β1,3-glucanase (Kapteyn et al., 1995), to release the β1,6-glucanase-resistant Pir-CWPs. In some experiments, Quantazyme-digested cell walls were further digested with exochitinase [0.3 U g−1 (wet weight of cell walls)] as has been described previously (Kollár et al., 1995). All enzymatic digestions were stopped by adding SDS at a final concentration of 0.4% (w/v) and heating for 3 min at 100°C.
The efficacy of the various hydrolytic enzymes to solubilize CWPs was studied by extracting isolated walls from cells that had been radiolabelled with [14C]-protein hydrolysate according to the procedure described by Kapteyn et al. (1997). In brief, cells were precultured in SC medium to OD600 = 1.0 (equivalent to 107 cells ml−1). Cells were collected, washed and resuspended in 100 ml of labelling medium, containing 0.17% (w/v) yeast nitrogen base without amino acids or ammonium sulphate (Difco), 2% (w/v) glucose, 0.05% (w/v) casein hydrolysate, 0.5% (w/v) ammonium sulphate, 0.25% (w/v) succinate and 0.001% (w/v) uracil with an initial density of 107 cells ml−1, and were incubated for 15 min at 28°C. Subsequently, [14C]-protein hydrolysate (0.46 Mbq; Amersham) was added, and the cultures were grown for 3.5 h at 28°C. Labelled cells were collected and homogenized as indicated above. Cell walls were isolated, SDS extracted, rinsed and digested with hydrolytic enzymes as described above. Protein contents of the different fractions were calculated as percentages of total label incorporated into SDS-extracted walls.
Enzymatic and chemical treatments of CWPs
β1,6-Glucanase-resistant CWPs released by Quantazyme were treated very mildly with alkali according to Kapteyn et al. (1999a). In brief, Quantazyme digests were dialysed, freeze-dried and incubated for 4 h at 4°C in 15 mM NaOH. The incubation was terminated by addition of neutralizing amounts of acetic acid, and alkali-treated CWPs were precipitated by addition of nine volumes of ice-cold methanol. After 2 h on ice, the sample was centrifuged at 14 000 g for 15 min, and the pellet was taken up in sample buffer as described by Laemmli (1970). Part of β1,6-glucanase-released and Quantazyme-released CWPs were treated with Endo-N-acetylglucosaminidase H (Endo H) (Boehringer Mannheim). CWPs dissolved in 50 ml of distilled water, containing 0.4% (w/v) SDS and 40 mM β-mercaptoethanol, were heated for 5 min at 100°C and were mixed with 50 mM sodium acetate, pH 5.5, containing 2.5 mM EDTA (150 ml). De-N-glycosylation was performed by adding 100 mU of Endo H. After incubating at 37°C for 17 h, Endo H-treated CWPs were precipitated by adding nine volumes of ice-cold methanol and were prepared for SDS–PAGE as described above. In some experiments, Quantazyme-resistant exochitinase-solubilized CWPs were digested with β1,6-glucanase according to the procedure described by Kapteyn et al. (1996).
Analysis of cell wall proteins
CWPs were separated by electrophoresis using linear 2.2–20% polyacrylamide gels and were electrophoretically transferred onto an Immobilon polyvinylidene difluoride (PVDF) membrane (Montijn et al., 1994). CWPs were visualized by probing the membranes with peroxidase-labelled Concanavalin A (ConA; 1 mg ml−1) in PBS, containing 3% (w/v) BSA, 2.5 mM CaCl2 and 2.5 mM MnCl2 (Klis et al., 1998). Western immunoblot analyses were performed with affinity-purified polyclonal antisera specifically directed against β1,6-glucan, and β1,3-glucan as described previously (Kapteyn et al., 1997; Klis et al., 1998). Blots were also probed with an antiserum raised against yeast Pir2p (Russo et al., 1992) according to Kapteyn et al. (1999a). Furthermore, we probed the PVDF filters with an anti-Als antiserum (Hoyer et al., 1998a). In this case, a serum dilution of 1:5000 in PBS, containing 5% (w/v) non-fat milk powder, was used, and before the blocking step the membranes were treated for 30 min with 50 mM periodic acid and 100 mM sodium acetate (pH 4.5) to enhance the specificity of the antiserum. In all cases, binding of antibodies was assessed with goat anti-rabbit IgG peroxidase (Pierce) at a dilution of 1:10 000 in PBS/5% (w/v) milk powder. The blots were visualized using ECL Western blotting detection reagents (Amersham) according to the manufacturer's instructions.
Determination of chitin content in cell walls
SDS-extracted cell walls (about 4 mg dry weight) were hydrolysed in 6 N HCl (1 ml) at 100°C for 17 h. After evaporation at 65°C, the samples were taken up in water (1 ml). A 0.1 ml volume of solution A (1.5 N Na2CO3 in 4% acetylacetone) was added to 0.1 ml of sample. The mixture was incubated at 100°C for 20 min, and 0.7 ml of 96% ethanol was added. One hour after adding 0.1 ml of solution B (1.6 g of p-dimethylaminobenzaldehyde in 30 ml of concentrated HCl and 30 ml of ethanol), the absorbance at 520 nm was measured and compared with a standard curve of 0–200 mg of glucosamine (Tracey, 1956; Popolo et al., 1997).
We thank Isaac Bom (Unilever Research Laboratories, Vlaardingen, The Netherlands) and Enrico Cabib (Laboratory of Biochemistry and Genetics, NIDDKD, MD, USA) for giving samples of pure endo-β1,6-glucanase and exochitinase respectively. We are grateful to Susan Southard (Center for Cancer Research, MIT, MA, USA) and Hisafumi Yamada-Okabe (Nippon Roche Research Center, Kamakura, Japan) for gifts of the C. albicans strains SSCA-2 and 211 respectively. We thank George Livi and Megan McLaughlin (SmithKline Beecham Pharmaceuticals) for donating the anti-Als antiserum. We also thank Joachim Ernst (Institut für Mikrobiologie, Heinrich-Heine-Universität Düsseldorf, Germany) for donating C. albicans strain CAP1-312 and for critical reading of the manuscript. Piet de Groot is thanked for his help with the homology searches of S. cerevisiae PIR genes with sequences of the Candida genome data bank. This work was financially supported by the Netherlands Technology Foundation (STW) and was co-ordinated by the Earth Life Sciences Foundation (ALW). L.L.H. was supported by Public Health Service Grant AI39441.