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Characterization and functional analysis of the β-1,3-glucanosyltransferase 3 of the human pathogenic fungus Paracoccidioides brasiliensis

Nadya Da Silva Castro, Kelly Pacheco De Castro, Ivan Orlandi, Luciano dos Santos Feitosa, Lívia Kmetzsch Rosa e Silva, Marilene Henning Vainstein, Sônia Nair Báo, Marina Vai, Célia Maria De Almeida Soares
DOI: http://dx.doi.org/10.1111/j.1567-1364.2008.00463.x 103-114 First published online: 1 February 2009


The fungus Paracoccidioides brasiliensis causes paracoccidioidomycosis, a systemic granulomatous mycosis prevalent in Latin America. In an effort to elucidate the molecular mechanisms involved in fungus cell wall assembly and morphogenesis, β-1,3-glucanosyltransferase 3 (PbGel3p) is presented here. PbGel3p presented functional similarity to the glucan-elongating/glycophospholipid-anchored surface/pH-regulated /essential for pseudohyphal development protein families, which are involved in fungal cell wall biosynthesis and morphogenesis. The full-length cDNA and gene were obtained. Southern blot and in silico analysis suggested that there is one copy of the gene in P. brasiliensis. The recombinant PbGel3p was overexpressed in Escherichia coli, and a polyclonal antibody was obtained. The PbGEL3 mRNA, as well as the protein, was detected at the highest level in the mycelium phase. The protein was immunolocalized at the surface in both the mycelium and the yeast phases. We addressed the potential role of PbGel3p in cell wall biosynthesis and morphogenesis by assessing its ability to rescue the phenotype of the Saccharomyces cerevisiae gas1Δ mutant. The results indicated that PbGel3p is a cell wall-associated protein that probably works as a β-1,3-glucan elongase capable of mediating fungal cell wall integrity.

  • Paracoccidioides brasiliensis
  • β-1,3-glucanosyltransferase
  • glycosylphosphatidylinositol anchored
  • cell wall biosynthesis


Paracoccidioides brasiliensis is a thermally dimorphic fungus that causes paracoccidioidomycosis, a systemic mycosis with a broad distribution in Latin America. Mycelia airbone propagules are inhaled and converted to the yeast form in the host lung, establishing the infection. The disease presents diverse clinical forms, ranging from asymptomatic pulmonary to severely disseminated and lethal infection (Restrepo, 2001).

The cell wall plays an essential role in the pathobiology of P. brasiliensis, because it is directly linked to the morphogenetic changes during phase transition. The fungal growth requires continuous remodeling of the cell wall polysaccharide network. Mycelium to yeast transition is characterized by a threefold increase in chitin content, as well as by a change of glucose polymer glucoside bonds, arranged only as β-1,3-glucan in the mycelium and mainly as α-1,3-glucan in the pathogenic yeast form (San-Blas & San-Blas, 1977). In this respect, studies on the fungal cell wall as well as the enzymes involved in cell wall biosynthesis and recycling provide excellent information for the design of antifungal drugs and for new preventive approaches.

Despite all the information accumulated about the cell wall structure, the enzymes responsible for its remodeling are still largely unknown, especially for P. brasiliensis. Several evidences suggest that for glucan-elongating protein (Gel), through its β-1,3-glucanosyltransferase activity, plays a role in the cross-linking of cell wall components in fungi (Popolo & Vai, 1999; Mouyna, 2000a). Gel1p of Aspergillus fumigatus (AfGel1p) catalyzes in vitro a two-step β-1,3-glucanosyltransferase reaction: (1) first, an internal glycosidic linkage of a donor β-1,3-glucan chain is cleaved and the reducing portion is released, (2) then the new reducing end is transferred to the nonreducing end of an acceptor β-1,3-glucan chain. Therefore, the generation of a new β-1,3-linkage between the acceptor and the donor molecule results in the elongation of β-1,3-glucan on branching points of other glucans, creating multiple anchoring sites for mannoproteins, chitin or for galactomannans. Thus, β-1,3-glucanosyltransferases act in a manner similar to glycoside hydrolases (GHs) in the first step, but a carbohydrate is preferred over a water molecule in the second one (Mouyna, 2000a, b, 2005; Ragni, 2007b). Despite the role of AfGel1p in the cell wall architecture, as mentioned above, no descriptions are found for Gel3p.

Gelp(s) are homologous to the glycophospholipid-anchored surface (Gas)/pH-regulated (Phr)/essential for pseudohyphal development (Epd), which are present in yeast species, fungi and human fungal pathogens (Saporito-Irwin, 1995; Mühlschlegel & Fonzi, 1997; Nakazawa, 1998, 2000; Mouyna, 2005; Ragni, 2007b), but not in mammalian cells (Klis, 1994). All these members are clustered in the GH72 family in the carbohydrate active enzymes database (CAZy) of GHs (http://www.cazy.org/fam/GH72.html).

The ScGas1, CaPhr1 and CaPhr2 proteins of Saccharomyces cerevisiae and Candida albicans, respectively, and more recently AfGel2p, a paralogue of AfGel1p, share the same catalytic residues and exhibit the same in vitro activity (Mouyna, 2000a, b, 2005). Moreover, AfGel1, AfGel2 and CaPhr1 restore the defective phenotype of an S. cerevisiae gas1 null mutant, indicating that these homologues are not only structurally but also functionally similar (Vai, 1996; Mouyna, 2000a, 2005). These proteins not only play an active role in the biosynthesis and morphogenesis by the correct incorporation of glucan molecules into the cell wall but are also required for virulence in C. albicans and A. fumigatus in a murine model of infection (Ghannoum, 1995; Mouyna, 2005), as well as in Fusarium oxysporum during plant infection (Caracuel, 2005).

In this paper, we described the first functional study of a Gel3p in pathogenic fungi. Our results indicated that PbGel3p is more abundant in the mycelium phase and is associated with the fungus cellular surface. Genetic complementation studies with the S. cerevisiae gas1 mutant demonstrated that PbGel3p is able to participate in the maintenance of fungal cell wall integrity by its ability to restore the Gas1p activity.

Materials and methods

Fungal strains and growth conditions

Paracoccidioides brasiliensis, isolate Pb01 (ATCC-MYA-826), which is standard to the studies in our laboratory (Bastos, 2007), and isolate from armadillo (PbAr) previously identified by Bagagli (2003) were used in this work. It was grown in semisolid Sabouraud medium as mycelium or yeast at 22 and 36 °C, respectively. The cells were maintained in liquid Sabouraud medium for 18 h before varying the culture temperature from 22 to 36 °C for the mycelium to yeast transition, which was maintained for 24 h.

The S. cerevisiae null mutant WB2d (gas1Δ∷LEU2), a derivative of W303-1B (MATαade2-1 his3-11,15 leu2-3,112 trp1-1 ura3-1 can1-100), was constructed in a previous study (Vai, 1996) and was the host strain for complementation experiments. Yeast cells were grown in batches at 30 °C in Difco yeast nitrogen base medium without amino acids (YNB−aa, 6.7 g L−1) containing glucose or galactose at 2% (w/v) and the required supplements. Buffered media were prepared by adding MES (10 g L−1) to YNB medium, followed by pH adjustment to 5.5 or 6.5. During growth, the pH was monitored and never varied by more than 0.1. Cell number was determined on mildly sonicated and diluted samples using a Coulter counter particle count and size analyser, model Z2, as described previously (Vanoni, 1983). Specific growth rates and duplication times (Td) were obtained by fitting the cell number against time.

Recombinant DNA procedures and plasmids

Paracoccidioides brasiliensis yeast cells were harvested, washed and frozen in liquid nitrogen. Grinding with a mortar and pestle broke the cells, and the genomic DNA was prepared by the cationic hexadecyl trimethyl ammonium bromide method according to Del Sal (1989). Paracoccidioides brasiliensis genomic DNA was used as a template for the PCR amplification of a partial fragment encoding the PbGel3p. The Gel3-S-1 (5′-CATCGATACCCTTGCCCCTTAC-3′) and Gel3-AS-1 (5′-CATAGATATTTGTTTGGGGTTGG-3′) oligonucleotide primers were designed based on the partial PbGEL3 sequence found in the P. brasiliensis ESTs available at the GenBank database (Felipe, 2003). The PCR reaction was conducted in a total volume of 25 μL containing 20 ng of DNA as a template. The resulting 752-bp product was subcloned into pGEM-T-Easy (Promega) and sequenced.

Southern blot analysis was performed on total DNA (25 μg) digested with the restriction enzymes XhoI, DraI, EcoRV, HindIII and SalI. Standard conditions for electrophoresis were used (Sambrook & Russell, 2001). The blot was probed to the 752-bp PbGEL3 genomic fragment labeled using the Gene Images Random Prime labeling module (GE Healthcare) and washed under high-stringency conditions according to the manufacturer's instructions. Hybridization was detected by a Gene Image CDP-Star detection module (GE Healthcare).

Cloning of the cDNA and genomic sequences encoding PbGel3p

A P. brasiliensis yeast phase cDNA library was constructed into EcoRI/XhoI sites of λ Zap II (Stratagene, LaJolla, CA) (Felipe, 2003). The screening of this library was performed using the 752-bp PCR fragment radiolabeled with [α-32P]dCTP. Plating 5 × 106 PFU, DNA transfer to membranes and hybridization were performed as described in standard procedures (Sambrook & Russell, 2001). Three positive clones were obtained and phage particles were released from the plaques. The in vivo excision of pBluescript phagemids (Stratagene) in Escherichia coli XL1 blue Minus Restriction (MRFs) was performed.

The PbGEL3 complete genomic sequence was obtained by PCR amplification of the total DNA of P. brasiliensis. The Gel3-S-2 (5′-CTCTCCAACCTCTCCAACCTCTCTC-3′) and Gel3-AS-2 (5′-ACACAATCACATCCCCTCCATCTCAC-3′) primers were constructed based on the cDNA sequence. The PCR reaction was performed with 20 ng of total DNA of P. brasiliensis. An amplified PCR product of 2199 bp was gel purified, subcloned into pGEM-T-Easy vector (Promega) and sequenced.

DNA sequencing and sequence analysis

The nucleotide sequences were determined on both strands by the dideoxy chain terminator method using a MegaBace® 1000 sequencer (GE Healthcare). The obtained sequences were translated and compared with all nonredundant polypeptides in the translated GenBank (http://www.ncbi.nlm.nih.gov) database. Amino acid analyses were performed using the PROSITE (http://us.expasy.org/prosite), PSORT II (http://www.psort.org/) databases and the big-PI fungal predictor (http://mendel.imp.univie.ac.at/gpi/fungi/gpi_fungi.html) (Eisenhaber, 2004) algorithm. The GenBank/EMBL/DDBJ accession numbers for the PbGEL3 sequences reported in this paper are AY324033 (cDNA) and DQ534494 (genomic).

Quantitative real-time PCR (QRT-PCR)

Total RNA of P. brasiliensis mycelium, mycelium during transition to yeast and yeast cells of Pb01 and P. brasiliensis isolated from armadillo (PbAr) was obtained. The fungal cells were harvested and frozen in liquid nitrogen, followed by grinding with a mortar and pestle. After addition of glass beads, the RNAs were extracted using Trizol (Invitrogen) according to the manufacturer's instructions. The quality of RNA was assessed using the A260 nm/A280 nm ratio, and by visualization of rRNA on 1.2% (w/v) agarose gel electrophoresis. In this case, a densitometric analysis of the ethidium bromide-stained bands of the different rRNA species was performed and the rRNA was calculated. The larger species was more intense than the 18S species, as described by Uppuluri (2007). The RNAs were used to construct single-stranded cDNAs using a reverse transcription system (Promega) following the recommendations of the manufacturer. As a control for genomic contamination, the same reactions were performed in the absence or presence of reverse transcriptase.

RNAs from P. brasiliensis were extracted and first-strand cDNAs were synthesized as described above. QRT-PCR reactions were performed in an ABI PRISM 7500 Sequence Detection System. The PCR thermal cycling conditions were as follows: an initial step at 50 °C for 2 min, followed by 5 min at 95 °C, and 40 cycles at 95 °C for 15 s, 60 °C for 10 s and 72 °C for 15 s. The Platinum SYBR Green qPCR Supermix (Invitrogen) was used as a reaction mixture, with addition of 10 pmol of each primer and 1 μL of template cDNA, in a final volume of 25 μL. Each cDNA sample was analyzed in triplicate with each primer pair. A melting curve analysis was performed at the end of the reaction to confirm a single PCR product. The data were normalized with ribosomal protein L34 (Andrade, 2006; Bastos, 2007) and ribosomal protein S30 cDNAs amplified in each set of QRT-PCR experiments. No statistical difference between these two normalizers was observed. Accordingly, the calibrator gene considered for the expression experiments was the one encoding L34 protein. The relative expression data were obtained using the 2−ΔΔCT method (Livak & Schmittgen, 2001). A nontemplate control with no genetic material was included to eliminate contamination or nonspecific reactions. The QRT-PCR primers for each gene were as follows: Gel3, 5′-CGTTGTCAGCGGAGGTATCGTC-3′ and 5′-AGGGCAGGTTCGGAGTTCAGTG-3′; L34, 5′- CGGCAACCTCAGATACCTTC-3′ and 5′-GGAGACCTGGGAGTATTCACG-3′.

Expression and purification of recombinant PbGel3p

The cDNA of PbGEL3 that encodes amino acids 19–529 (predicted mature protein; see Fig. 1) was subcloned into the pGEX-4T-3 (GE Healthcare). EcoRI/XhoI restriction sites (underlined) were introduced into the Gel3-S-3 (5-GAATTCCGCTGACCTGGATCCTATTGTC-3′) and Gel3-AS-3 (5′-CTCGAGTTACAACAACAAAATACTCATC-3′) oligonucleotides for the DNA synthesis. The obtained plasmid was sequenced in both strands and used to transform E. coli BL21 pLysS. The recombinant P. brasiliensis Gel3 protein (rPbGel3p) was induced with 0.1 mM IPTG and purified by affinity chromatography, as reported previously (Castro, 2008).


Comparison of the deduced amino acid sequence of PbGel3p with those of β-1,3-glucanosyltransferases from fungi. Asterisks indicate amino acid identity and dots represent conserved substitutions. The hydrophobic amino and carboxy termini are indicated by white letters and black blocks. Conserved catalytic motifs are boxed (dotted lines) and the conserved glutamate residue into the catalytic site is detached by italic letters. Fourteen aligned cysteine residues are indicated by arrows and bold letters. Predicted glycosylphosphatidylinositol anchor sites in the C-terminal regions are boxed (solid lines). The GH72 and Cys-Box domains are indicated above the amino acid sequences. Accession numbers were as follows: Ajellomyces capsulatus (AcGel3, XP_001539818); Aspergillus terreus (AtEpd1, XP_001212455); Aspergillus fumigatus (AfGel3, AAF40140); and Saccharomyces cerevisiae (ScGas1, CAA89140).

Antibody production

Polyacrylamide gel containing 100 μg of the rPbGel3p was injected into rabbit three times at 10-day intervals to generate specific rabbit polyclonal serum. Both rabbit preimmune and immune serum (containing anti-rPbGel3p polyclonal antibody) were sampled and stored at −20 °C.

Western blotting analysis

Paracoccidioides brasiliensis protein extracts were obtained by disruption of frozen cells in the presence of protease inhibitors. The mixture was centrifuged at 12 000 g at 4 °C for 15 min, and the supernatant was used. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was carried out and the proteins (30 μg) were electrophoretically transferred to a nylon membrane, according to standard protocols. PbGel3p was detected with the polyclonal antibody (1 : 1000 diluted). The reaction was revealed with 5-bromo-4-chloro-3-indolylphosphate/nitroblue tetrazolium. Negative controls were obtained with rabbit preimmune serum.

Protein extracts from S. cerevisiae cells, prepared as described in (Mouyna, 2005), were resolved by SDS-PAGE on 8% polyacrylamide gels. After blotting, filters were stained for total protein with Ponceau Red (Sigma) before immunolabeling, which was performed using anti-PbGel3p antibody (1 : 1000 diluted). Binding was visualized with the ECL Western Blotting Detection Reagents (GE Healthcare).

Confocal analysis

The cellular localization of the PbGel3p was performed as described by Batista (2006). Images of diamidino-2-phenylindole-stained cells were observed in a Bio-Rad 1024 UV confocal system attached to a Zeiss Axiovert 100 microscope, using a × 40 numerical aperture, a 1.2 plan-apochromatic differential interference contrast water immersion objective. All images were collected by Kalman averaging at least every eight frames (512 × 512 pixels), using an aperture (pinhole) of 2 mm.

Transmission electron microscopy of P. brasiliensis yeast cells and immunocytochemistry of the Gel3p

For the ultrastructural and immunocytochemistry studies, we used the protocols described previously in Barbosa (2006). The ultrathin sections were incubated with the polyclonal antibody to the rPbGel3p (diluted 1 : 100), washed and then incubated with the labeled secondary antibody (anti-mouse IgG, Au conjugated, 10 nm average size; 1 : 20 diluted). The grids were observed with a Jeol 1011 transmission electron microscope (Jeol, Tokyo, Japan). Controls were incubated with mouse preimmune serum (1 : 100 diluted).

Saccharomyces cerevisiae genetic procedures

For ectopic expression of P. brasiliensis PbGEL3 in S. cerevisiae, a 2-kb HindIII/XhoI fragment containing the PbGEL3 cDNA and its 5′ and 3′ untranslated regions was cloned into HindIII/XhoI-digested pYES2 (Invitrogen) under the control of the GAL1 promoter. The resulting plasmid was used to transform the WB2d strain. The transformed strain is indicated throughout the text as YGEL3. For ectopic expression of S. cerevisiae GAS4, the whole coding sequence of GAS4 plus 203 bases downstream was PCR-amplified from yeast chromosomal DNA and fused with the PCR-amplified promoter region of S. cerevisiae GAS1 to obtain expression in vegetative growth. PCR amplifications were carried out using specific primers where appropriate restriction sites were introduced at the extremes to facilitate fusion and cloning. PCR products were first subcloned into the pGEM-7Zf(+) (Promega), generating the pG4 plasmid, and then into the high-copy YEp24 vector. The latter resulting vector was introduced into WB2d, generating the YGAS4 strain. PCR products were routinely checked by sequence analysis. The sequences of all the oligonucleotide primers used for PCR are available upon request. Standard methods were used for DNA manipulation and yeast transformation (Hill, 1991; Sambrook & Russell, 2001).

Light and fluorescence microscopy

Saccharomyces cerevisiae cellular morphology was examined by Nomarski phase-contrast microscopy. Chitin was visualized after staining with calcofluor white (CW; Fluorescent Brightener 28, Sigma-Aldrich) as reported (Cipollina, 2007) under a Nikon Eclipse E600 fluorescence microscope equipped with a Leica DC 350F ccd camera.

Statistical analysis

Duplication time values are presented as means ± SDs. Pairwise comparisons between duplication times were made for mean values of the different strains using Student's t-test. The level of statistical significance was set at a P value of≤0.01.

Sensitivity assay

To determine the sensitivity of the different strains to SDS and to CW, yeast cells exponentially growing in galactose medium (pH 6.5) were dropped (5 μL from a concentrated suspension of 107 cell mL−1 and from serial 10-fold dilutions) onto galactose (pH 6.5) medium plates supplemented with 0.01% (w/v) SDS or 50 μg mL−1 of CW. Plates were incubated at 30 °C for 3 days. Cells were also dropped onto plates without SDS and CW to monitor cell growth.


Isolation and sequence analysis of PbGEL3

A blastx search of the partial P. brasiliensis EST available in the GenBank database (Castro, 2005) revealed an 812-bp ORF that showed a high sequence homology (51% identity, 66% similarity) to Aspergillus nidulansβ-1,3-glucanosyltransferase 3 (Mouyna, 2000b). In order to isolate the complete cDNA encoding PbGel3p, we initially obtained a genomic PCR product corresponding to the described ORF and used it as a probe to screen a yeast P. brasiliensis cDNA library. The entire cDNA consisted of 2057 bp and encoded a 529-amino-acid polypeptide with a theoretical molecular mass of 57.1 kDa and a pI of 6.1. The complete genomic sequence was obtained by PCR amplification using oligonucleotide primers complementary to the cDNA sequence. The PbGEL3 included four introns of 79, 133, 78 and 68 bp (data not shown).

The deduced amino acid sequence of PbGEL3 showed significant homology to Gel3p and all four homologues, Gel/Gas/Phr/Epd, belonging to the GH72 family. The highest sequence similarity and identity were among the Gel3 proteins of P. brasiliensis, Ajellomyces capsulatus, Aspergillus terreus and A. fumigatus (Fig. 1) and were most evident within the region of the first 300 amino acids. The PSORT analysis revealed a putative N-terminal signal peptide, with a predicted cleavage site between amino acids 18 and 19 (Fig. 1). The position of the 14 cysteine residues was conserved among the Gel3 proteins shown in Fig. 1. A domain named Cys-Box, which contained six conserved cysteine residues and is present in some members of the GH72 family, was also found in PbGel3p near the carboxy-terminal region. Two glutamic residues (E159 and E260), essential for the enzymatic activity, were conserved in the catalytic domain. The potential glycosylphosphatidylinositol anchor site at S498 and a hydrophobic region encompassing residues 512–528 (VGAGVVAGVIAGMSILL) were found at the carboxy terminus (Fig. 1).

Hybridization analysis

Southern blot analysis using a 752-bp PbGEL3 probe under high-stringency conditions was able to detect a single DNA fragment in the P. brasiliensis DNA digested with the restriction enzymes XhoI, DraI, EcoRV, HindIII and SalI (Fig. 2a, lanes 1, 2, 4 and 5, respectively). EcoRV digestion produced fragments consistent, in number and size, with the single restriction site presumed to occur in PbGEL3 (Fig. 2a, lane 3). The obtained restriction profiles indicated that the P. brasiliensis genome contained a single copy of the PbGEL3 gene. Confirming this suggestion, one copy of the PbGEL3 gene was detected by in silico analysis at the genome project developed by BROAD Institute (http://www.broad.mit.edu/annotation/genome/paracoccidioides_brasiliensis/MultiHome.html).


Analysis of the Paracoccidioides brasiliensis GEL3 gene organization and evaluation of the expression levels. (a) Southern blot analysis of PbGEL3. Total DNA (25 μg) was digested with restriction enzymes XhoI, DraI, EcoRV, HindIII and SalI (lanes 1–5, respectively). The blot was hybridized to a 752-bp PbGEL3 labeled PCR fragment. (b) QRT-PCR plot of PbGEL3 expression levels in different phases and isolates. cDNAs derived from mycelium (M), mycelium during 24 h of transition to yeast (T) and yeast (Y) cells of two P. brasiliensis isolates, Pb01 and PbAr, are shown. The ΔΔCt method was used to calculate the relative amount of specific RNA present in each sample relative to the yeast phase of the Pb01 isolate (set as 1.0). The L34 gene was used to normalize each reaction. (c) Western blot analysis of the native PbGel3p in a cellular extract during the dimorphic transition. The samples from mycelium (M), mycelium during 24 h of transition (T) and yeast (Y) cells were fractionated (12% SDS-PAGE) and transferred to a membrane. The blots were reacted to the rabbit polyclonal anti-rPbGel3p antibody and developed with 5-bromo-4-chloro-3-indolylphosphate/nitroblue tetrazolium. Molecular size markers are indicated.

Transcript analysis by QRT-PCR

Template cDNAs derived from mycelium (M), mycelium during transition to yeast (T) and yeast (Y) cells of the Pb01 isolate and of PbAr isolate were used to estimate the relative transcript levels of the PbGEL3 by QRT-PCR analysis. The L34 mRNA was chosen as a reference transcript, because the expression of the L34 gene does not fluctuate significantly during the differentiation of P. brasiliensis (Andrade, 2006; Bastos, 2007). As shown in Fig. 2b, a peak of expression occurred in the mycelium (M) phase, which decreased considerably during 24 h of transition (T), reaching a faint expression in the yeast (Y) phase in the Pb01 isolate. The mycelium and transition cells revealed an increase in the transcript level of 104.9- and 8.6-fold, respectively, compared with yeast cells (Fig. 2b). The transcript was also present at low levels in the yeast phase of the PbAr isolate (Fig. 2b).

Expression, purification and detection of Gel3p in P. brasiliensis

The cDNA encoding the P. brasiliensis Gel3p was subcloned into the expression vector pGEX-4T-3 to obtain the recombinant fusion protein that was purified by affinity chromatography and cleaved by addition of thrombin (data not shown). Protein extracts from mycelium (M), mycelium during 24 h of transition to the yeast phase (T) and yeast (Y) cells were blotted onto nitrocellulose membranes and reacted to the polyclonal antibody (Fig. 2c). As demonstrated, a single band of 58 kDa was detected in all extracts. The protein is strongly accumulated in the mycelia phase (Fig. 2c) and its expression is decreased during the transition to yeast (Fig. 2c). No cross-reactivity to the rabbit preimmune serum was evidenced with the samples (data not shown).

Determination of PbGel3p cellular localization by confocal and immunoelectron microscopy analysis

Representative confocal microscopy images of mycelium (Fig. 3a–d) and yeast (Fig. 3e–h) cells of P. brasiliensis showed that anti-rPbGel3p reacted with the surface and cytoplasmatic components of both phases (Fig. 3d and h). The cell surface fluorescence was clearer in yeast cells (Fig. 3h) due to their definite rounding format and larger size. No cross-reaction was observed with the preimmune serum (Fig. 3b and f).


Distribution of Gel3p in Paracoccidioides brasiliensis. (a, c) Mycelium and (e, g) yeast cells using differential interferential contrast microscopy. (b, f) Control systems, without polyclonal antibodies before incubation with fluorescein isothiocyanate-labeled rabbit anti-IgG. Confocal microscopy with antibodies generated against the recombinant GST-PbGel3p in (d) mycelium and (h) yeast cells. Labeling of P. brasiliensis was obtained by anti-rPbGel3p polyclonal antibodies (green). Scale bar=10 μm.

In order to detail the cellular localization of PbGel3p, we further performed immunocytochemistry experiments in yeast cells. The cell surface and organelle structures were preserved and free of label when incubated with the rabbit preimmune serum (Fig. 4a). In yeast cells processed by the postembedding method, gold particles were predominantly associated with the cell wall (Fig. 4b and c). The number of gold-labeled particles, counted in five immunocytochemistry assays, was significantly higher in the cell wall (P<0.05) than in the cytoplasmic compartment (data not shown).


Immunoelectron microscopy detection of Gel3p in Paracoccidioides brasiliensis yeast cells by postembedding methods. (a) Negative control exposed to the rabbit preimmune serum in transmission electron microscopy of P. brasiliensis yeast cells. (b and c) Gold particles are observed at the fungus cell wall (arrow) and in the cytoplasm (double arrowheads). n, nucleus; v, intracytoplasmic vacuoles; m, mitochondria; w, cell wall.

PbGEL3 completely suppresses the S. cerevisiae gas1 disruptant phenotype at pH values above 5

The PbGEL3 cDNA was expressed, under the control of the GAL1 promoter, in the gas1Δ background and the resulting phenotype was analyzed in galactose-containing medium (inducing condition). Total proteins from the transformed strain were analyzed by immunoblot using the antibody against PbGel3p. In these cells, a unique band of about 58 kDa was detected that was absent in untranformed cells (Fig. 5a). Moreover, as shown in Table 1, PbGEL3 partially suppressed the slow growth of gas1Δ cells. In fact, cells expressing the PbGEL3 cDNA (YGEL3 strain) had a Td of about 2.31 h, an intermediate value between those determined for W303-1B and gas1Δ-empty vector strains (Td of 1.83 and 3.02 h, respectively). A partial reversion was also observed for other phenotypic traits such as abnormal morphology and CW sensitivity (data not shown).


Paracoccidioides brasiliensis Gel3p restores the phenotypic defects of the Saccharomyces cerevisiae gas1Δ mutant at pH 6.5. (a) Total extracts from S. cerevisiae cells exponentially growing in unbuffered and buffered media were analyzed by immunoblotting with anti-PbGel3p antibodies. The same amount of proteins (70 μg) was loaded on each lane. A sample from mycelium of P. brasiliensis was also loaded as a control. (b) Cellular morphology visualized by Nomarsky (upper panel) and CW staining (lower panel) of exponentially growing yeast cells in galactose medium, pH 6.5. Five microliters from a concentrated suspension (107 mL−1) of yeast cells grown as in (b) and from serial 10-fold dilutions were spotted onto galactose medium plates, pH 6.5, with or without CW (c) and with or without SDS (d). WT, wild type.

View this table:

Paracoccidioides brasiliensis GEL3 abrogates the slow growth phenotype of Saccharomyces cerevisiae gas1Δ mutant according to the pH of the growth medium

Growth conditionStrainTd (h)
Galactose medium (unbuffered)W303-1B1.83 ± 0.08
gas1Δ3.02 ± 0.1
YGEL32.31 ± 0.06
YGAS42.33 ± 0.07
Galactose medium (pH 5.5)W303-1B2.33 ± 0.11
gas1Δ4.12 ± 0.12
YGEL32.37 ± 0.09
YGAS42.40 ± 0.12
Galactose medium (pH 6.5)W303-1B2.46 ± 0.07
gas1Δ4.15 ± 0.09
YGEL32.42 ± 0.11
YGAS42.43 ± 0.14
  • Student's t-test was applied for their comparison setting a P value of ≤0.01.

  • * The Td was calculated as ln2/k, where k is the constant rate of exponential growth. Data represent the average of three independent experiments; SDs are indicated.

We considered that the partial rescue of the gas1Δ mutant phenotype could be ascribed to nonoptimal environmental conditions for PbGel3p activity as reported recently for Gas2 and Gas4 proteins of S. cerevisiae, two redundant versions of Gas1p that are specialized to function at pH values close to neutrality (Ragni, 2007a). Thus, we analyzed the YGEL3 phenotype in galactose media buffered to pH 5.5 and 6.5 to avoid acidification that usually takes place following yeast cells' growth (Sigler & Höfer, 1991). At pH 6.5, in theYGEL3 strain, the protein of about 58–60 kDa showed levels comparable to those detected in the unbuffered growth medium (Fig. 5a). The same results were obtained for cells grown at pH 5.5 (data not shown). Interestingly, in both buffered media, the YGEL3 strain behaved in a manner similar to gas1Δ cells expressing GAS4 (YGAS4 strain). In fact, under these two growth conditions, PbGEL3, like ScGAS4, completely rescued growth rate defects of gas1Δ cells (Table 1), whose Td increased along with the increase in pH. In addition, YGEL3 cells reassumed the ellipsoidal shape and cells carrying two or more buds (pluribudded cells) that are distinctive of gas1Δ were absent (Fig. 5b). Moreover, after CW staining for chitin, YGEL3 cells showed definite fluorescence in the bud scars and at the mother–daughter junction. Chitin in cells deprived of Gas1p activity increased and was delocalized (Fig. 5b) as expected. In the gas1 null mutant, the increase of chitin level determined a hypersensitivity to growth in the presence of CW. Thus, cells exponentially growing in galactose medium (pH 6.5) were spotted onto plates of the same medium containing 50 μg mL−1 of CW or onto plates without it to monitor cell growth. PbGEL3 completely abolished the CW hypersensitivity of gas1Δ cells (Fig. 5c). Similar results were obtained for YGEL4 (data not shown). Finally, we tested the sensitivity to SDS. In fact, gas1Δ cells are also hypersensitive to growth in the presence of such an anionic destabilizing detergent, as a consequence of a weakened cell wall (Vai, 1996). As shown in Fig. 5d, YGEL3 displayed the same sensitivity to SDS as YGAS4 and the reference wild-type strains.


The mechanisms through which the cell wall proteins of P. brasiliensis respond to different environmental conditions remain largely unknown. In an effort to understand the mechanisms that are involved in this fungus cell wall assembly and integrity maintenance, we initially searched for cDNAs encoding homologues of cell wall-associated proteins (Castro, 2005). We identified an 812-pb fragment ORF that encodes a homologue of Gel3p of A. nidulans (Mouyna, 2000b). The full-length cDNA and gene were obtained. The complete PbGel3p deduced amino acid sequence presented homology to glycosylphosphatidylinositol-anchored Gas/Gel/Phr/Epd proteins, which are plasma membrane and cell wall-associated enzymes, responsible for the elongation of β-1,3-glucan chains and are required for correct morphogenesis in yeast (Mouyna, 2000a). All these proteins belong to the GH72 family (CAZy database), whose conformation of the catalytic domain is predicted to assume a TIM-barrel shape (Mouyna, 1998; Papaleo, 2006). These enzymes, which are organized in characteristic domains, contained basically: (1) a signal peptide and an N-terminal catalytic domain with two conserved glutamate residues identified as essential for catalysis and (2) a hydrophobic attachment signal characteristic of glycosylphosphatidylinositol-anchored proteins in the C-terminus region (Mouyna, 2000a, b; Carotti, 2004; Ragni, 2007b). Nevertheless, in some members of the GH72 family (subfamily 72+), including PbGel3p, a Cys-box domain can be found that contain six conserved cysteine residues presumably involved in the formation of three disulfide bridges (Palomares, 2003; Carotti, 2004). This domain is present in some β-1,3-glucanases of plants and it is associated with enzyme activity (Palomares, 2003; Barral, 2005). Moreover, a previous study revealed that the removal of this domain in S. cerevisiae Gas1p totally abolished the enzymatic activity, showing, therefore, that it is essential for the function and stability of the protein (Carotti, 2006).

QRT-PCR and Western blot analysis demonstrated the PbGel3p expression. The transcript and protein levels were more abundant in the mycelium phase. A drastic reduction in both the transcript and the protein was observed during the first 24 h of temperature shift from 22 to 36 °C, reaching low levels in the yeast phase. The mycelium cell wall of P. brasiliensis is composed mainly of β-1,3-glucan. In agreement, the PbGEL3 transcript and protein expression levels described here predominate largely in the mycelium phase. The decrease in expression as the fungus triggers the differentiation to yeast is consistent with transforming to α-glucan when the yeast phase is reached (San-Blas & San-Blas, 1994). The data also suggest a putative role of PbGel3p in cell wall maintenance and fungal morphology. In fact, this protein family has been described in the plasma membrane (Popolo, 1988; Fankhauser & Conzelmann, 1991), as well as covalently linked to the cell wall via a glycosylphosphatidylinositol remnant (De Sampaïo, 1999; Yin, 2005). The confocal and immunoelectron microscopy analysis confirmed the preferential surface localization of PbGel3p.

Although the absence of an efficient molecular toolbox for gene disruption in P. brasiliensis has limited the functional studies in the organism itself, we addressed the potential role of PbGel3p in cell wall biosynthesis and morphogenesis by investigating whether it could rescue in vivo the S. cerevisiae gas1Δ mutant phenotype. In fact, in S. cerevisiae the lack of Gas1 β-1,3-glucanosyltransferase activity drastically affects the correct relative proportion and the degree of cross-linking of cell wall constituents (Popolo, 1997; Ram, 1998). This leads to a detectable mutant phenotype characterized by reduced growth, round-cell morphologies, sensitivity to CW, increased cell wall permeability and secretion (Popolo & Vai, 1999; Vai, 2000). We observed that PbGEL3 was able to partially complement the mutant phenotype of gas1Δ in unbuffered medium and fully at higher pH values. Similar results were found for GAS4 of S. cerevisiae, a paralogue of GAS1, encoding a protein specialized to function at a pH value close to neutrality (Ragni, 2007a). In this growth condition, PbGEL3 was able to correct the alterations of cell wall constituents due to GAS1 inactivation, as observed by the ellipsoidal morphology and the Td similar to that of wild-type cells displayed by YGEL3 cells, by their ability to grow in the presence of CW and SDS and by the quantity and localization of chitin molecules. This indicates that PbGEL3 expression abrogates gas1Δ structural defects that depend on altered connections between glucans and mannoproteins, which are required to form a selective barrier at the outer surface of the cell wall. On the whole, our results show that PbGel3p is functional in S. cerevisiae and it suppresses both the morphological defects and the compensatory responses induced by the lack of Gas1p. The complete functionality of PbGel3p is detected at extracellular pH values above 5. This requirement could reflect both the environmental growth conditions of P. brasiliensis and the pH of the niches colonized by this dimorphic pathogen. In fact, yeast forms did not grow below pH 3.6; most of the clinical isolate strains require a pH of culture medium above 5.6 and the establishment of yeast infection takes place in host tissues at pH values close to neutrality (Franco, 1986; Sano, 1997; Restrepo, 2001).

Thus, our results suggest that, similar to Gas1p of S. cerevisiae, PbGel3p can play an active role in biosynthesis and morphogenesis of the P. brasiliensis cell wall, especially in mycelium cells, the fungus infective phase. Elucidation of these molecular mechanisms involved in the cell wall assembly of P. brasiliensis is important and necessary once the pathogenicity of this fungus appears to be correlated with changes in the cell wall composition, organization and structure that occur during the morphogenetic transition from the mycelium to the yeast form.


This investigation at Universidade Federal de Goiás was financially supported by CNPq (Grant no. 505658/2004-6), FINEP (Grant no. 0104077500 and 0106121200), FAPEG and SECTEC-GO. K.P.C. and N.S.C. have a fellowship from CAPES. We thank Gustavo A. Niño-Vega, IVIC, Venezuela, for providing the plasmid pYES2.


  • Editor: José Ruiz-Herrera


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