Biochemical Characterization of Paracoccidioides brasiliensis α-1,3-Glucanase Agn1p, and Its Functionality by Heterologous Expression in Schizosaccharomyces pombe

α-1,3-Glucan is present as the outermost layer of the cell wall in the pathogenic yeastlike (Y) form of Paracoccidioides brasiliensis. Based on experimental evidence, this polysaccharide has been proposed as a fungal virulence factor. To degrade α-1,3-glucan and allow remodeling of the cell wall, α-1,3-glucanase is required. Therefore, the study of this enzyme, its encoding gene, and regulatory mechanisms, might be of interest to understand the morphogenesis and virulence process in this fungus. A single gene, orthologous to other fungal α-1,3-glucanase genes, was identified in the Paracoccidioides genome, and labeled AGN1. Transcriptional levels of AGN1 and AGS1 (α-1,3-glucan synthase-encoding gene) increased sharply when the pathogenic Y phase was cultured in the presence of 5% horse serum, a reported booster for cell wall α-1,3-glucan synthesis in this fungus. To study the biochemical properties of P. brasiliensis Agn1p, the enzyme was heterologously overexpressed, purified, and its activity profile determined by means of the degradation of carboxymethyl α-1,3-glucan (SCMG, chemically modified from P. brasiliensis α-1,3-glucan), used as a soluble substrate for the enzymatic reaction. Inhibition assays, thin layer chromatography and enzymatic reactions with alternative substrates (dextran, starch, chitin, laminarin and cellulose), showed that Agn1p displays an endolytic cut pattern and high specificity for SCMG. Complementation of a Schizosaccharomyces pombe agn1Δ strain with the P. brasiliensis AGN1 gene restored the wild type phenotype, indicating functionality of the gene, suggesting a possible role of Agn1p in the remodeling of P. brasiliensis Y phase cell wall. Based on amino acid sequence, P. brasiliensis Agn1p, groups within the family 71 of fungal glycoside hydrolases (GH-71), showing similar biochemical characteristics to other members of this family. Also based on amino acid sequence alignments, we propose a subdivision of fungal GH-71 into at least five groups, for which specific conserved sequences can be identified.


Introduction
Paracoccidioidomycosis (PCM) is a human systemic mycosis caused by four species, comprised by the Paracoccidioides brasiliensis complex (S1, PS2 and PS3; [1]) and Paracoccidioides lutzii, a recently described species, so far reported only in Brasil [2]. Confined geographically to Latin America, where it is one of the most frequent systemic mycoses, PCM may result in a fatal outcome [3]. In P. brasiliensis, changes in cell wall composition associated to the thermal dimorphism exhibited by this fungus, are closely related to pathogenicity and virulence [4]. Experimental evidence suggests that P. brasiliensis cell wall a-glucan, the fungal outermost layer, plays a protective role against host defense mechanisms [5]. Later studies in Histoplasma capsulatum [6], confirmed San-Blas and San-Blas' findings [5] with regard to the importance of a-1,3-glucan as a virulence factor. Furthermore, the wide layer of a-1,3-glucan in H. capsulatum yeast cell wall hides the underlying b-1,3-glucan, preventing in this way its efficient exposure to macrophages, and impairing the secretion of TNFa. As a result, the immune response of the infected organism is reduced [7]. The absence of a-1,3glucan in mammalian cells, raises the possibility of developing specific antifungal drugs targeted toward the blockage of a-1,3glucan biosynthesis, which might result in depression of fungal virulence, allowing the natural immune response of the infected organism towards the fungus, and preventing the disease.
a-1,3-Glucanases (EC 3.2.1.59), also called mutanases due to their ability to degrade the extracellular glucan synthesized by the bacterium Streptococcus mutans [13], are enzymes capable of hydrolyzing glucose polymers linked by a-1,3 glycosidic bonds. According to their amino acid sequence, these enzymes are grouped into the family 71 of glycoside-hydrolases (GH-71). Depending on the final products, either oligo-or monosaccharides, they are divided into endolytic or exolytic enzymes [14].
In S. pombe, two a-1,3-glucanase genes are present (agn1 and agn2), whose translation products Agn1p and Agn2p are involved in different cell processes. Agn1p is involved in cytokinesis [15]. S. pombe agn1 mutants are unable to separate as free cells, impairing the physical division of the cell during cell fission [15,16]. Meanwhile, Agn2p is involved in the process of sexual differentiation, sporogenesis or spore formation, specifically in the process of ascospore release, as demonstrated by its inhibition in S. pombe agn2 mutants [17].
After the exhaustion of glucose, A. nidulans a-1,3-glucanase is secreted to the cell wall and mobilizes a-1,3-glucan, the main reserve material accumulated during vegetative growth in the cell wall; once monosaccharides are released, they are captured and metabolized by the cell during starvation [18]. In Trichoderma harzianum, a-1,3-glucanase degrades cell wall of plant pathogenic fungi, thus becoming an inhibitor of spore germination and mycelial growth of a wide range of fungal pathogens [19]. Additionally, in fungi the morphological changes associated with extensive alterations in cell wall composition are regulated by the action of polysaccharide synthases and hydrolases. These enzymes may facilitate the complex patterns of lysis, branching and crosslinking of glucans involved in the process of fungal wall synthesis.
As a further step into the comprehension of the cell wall a-1,3glucan metabolism in P. brasiliensis, we aimed to characterize the P. brasiliensis a-1,3-glucanase by heterologous expression of its encoding gene, AGN1, and purification of its transcriptional product, Agn1p. Functionality of the gene was assessed by complementation of an S. pombe agn1D mutant with the P. brasiliensis AGN1 gene.

Nucleic Acids Isolation
Genomic DNA (gDNA) extraction was performed as previously described [21]. RNA was obtained from freeze-dried macerated cells of P. brasiliensis using TRIzol Reagent TM (GIBCO Life Technologies, Rockville, USA), following the manufacturer's instructions. S. pombe gDNA from wild type strain wt-64, or plasmid DNA from S. pombe strain 1252, were isolated according to Hoffman and Winston [22]. The AxyPrep Multisource Total Miniprep Kit (Axygen Biosciences) was used for extraction of total RNA from the mutant strain 1252, following the manufacturer's recommendations.

Isolation and Sequencing of AGN1
For isolation of P. brasiliensis AGN1, a HindIII partial genomic library was constructed as follows: 100 mg of P. brasiliensis DNA were digested with HindIII (Invitrogen), and size-fractionated fragments (according to Southern analysis) were cloned into pBluescript SK vector (Stratagene). Resulting transformants were collected and screened by colony hybridization, with a 750 bp PCR amplified fragment of the putative H. capsulatum AGN1 gene, by using Mut(F): 59 ATY GAY GCA TTY GCW CTY AAY 39 and Mut (R): 59 GAY TCR CCG TAG TC 39, primers. A positive clone yielding plasmid pMP1, containing a 2.3 kb insert was isolated and sequenced, showing to contain a partial sequence of the gene. The complete P. brasiliensis AGN1 gene sequence was

Computer-assisted Sequence Analyses
Assembly of the nucleotide sequences and translated protein sequences were generated with the Vector NTI suit package (Vector NTI, InforMax, Inc., USA). Homology searches were performed on the GenBank database using BLAST 2.0 [23]. Domain analyses of Agn1p were performed using SMART internet service for sequence analyses and prediction of protein structure and function [24], identification of protein patterns and . The Mega 4 software package was employed, using ClustalW for sequence alignment. Construction of the phylogenetic tree was done by the neighbor-joining method using 1000 replications. Five groups (G1-G5) are distinguished. P brasiliensis Agn1p (labeled Pb73) is located in group G1. The groups are: G1 (red), G2 (blue), G3 (black), G4 (green), and G5 (purple). GenBank accession numbers of sequences, and names of fungal species used for construction of the tree are displayed in Table 2

Quantitative RT-PCR
Total RNA was treated with DNase by using the TURBO DNA free TM kit (Ambion Inc., Austin, TX, USA). The RETRO-Script TM kit (Ambion Inc., Austin, TX, USA) was used for reverse transcription of mRNA. For real-time PCR of AGN1, primers RT3:59-GCA GCA AGT TAT CAC ACT AC-39 and RT4:59-TGG TTC CGT CAT ACA TTT TA-39 were used. For expression analysis of AGS1, sequence specific primers FrwAGS1_RT: 59-AAA TGC GGC ACG GAG GAG A-39 and RevAGS1_RT: 59-AAG GGT GGT ATC AAG TGC CGA GT-39 were used. To find the best internal control as normalizer for the expression experiments, two genes were used. Amplification of 18S rRNA was carried out, using the primers 18S S3:59-CGA TTC CGG AGA GGG AGC C-39 and 18S AS3:59-CGT ATC GGG ATT GGG TAA TTT GC-39. A second reference gene (Pbl34) which has no changes in transcription on both morphologies [30] was also analyzed, using the primers designed by Moreira-Dantas [30]. In experiments aimed to evaluate the changes induced by horse serum, changes in expression levels of the 18S gene were observed. Therefore, the Pbl34 gene was chosen as the normalizer gene for all subsequent experiments. Quantitative PCR was performed in triplicate on an iQ5 real time PCR detection system, using the GoTaqH qPCR Master Mix (Promega Corporation, Madison, WI, EE.UU), in a 15 ml volume (7.5 ml Master Mix 2X, 5.5 ml of a forward and reverse primer mix 0.2 mM, and 2 ml cDNA). Reaction conditions were as follows: 95uC for 3 min, followed by 40 cycles at 94uC for 10 s, 58uC for 30 s, and 72uC for 30 s, with dissociation conditions of 95uC for 1 min, 55uC for 1 min, and 81 cycles starting at 55uC, with temperature increases of 0.5uC every 10 s up to 95uC. PCRs with serial dilutions of P. brasiliensis cDNA as template were used to  calculate the amplification efficiency for each pair of primers. All Ct values were normalized to the Ct values of the standard gene and the relative expression levels were calculated using the 2 2DDCT method [31]. Statistical analysis of the data was done by comparing their mean expression levels, using the Turkey-Kramer test, included in the InStat statistical package (GraphPad Software).

Extraction and Solubilization of a-1,3-glucan
In order to test the enzymatic activity and specificity of P. brasiliensis Agn1p, P. brasiliensis yeast-like cell wall was extracted by alkaline separation as in [36]. The alkali-insoluble a-1,3-glucan was converted into soluble carboxymethyl-a-1,3-glucan (SCMG) to ensure its availability in aqueous solution for enzymatic assays. For this, monochloroacetic acid was used in basic medium to modify the hydroxyl group of carbon 6 [37,38]. Briefly, 318 mg of a-1,3-glucan were resuspended in 10 ml 2-propanol with stirring for 30 min at room temperature. Next, 0.5 ml of 30%NaOH was added dropwise for 60 min with agitation. The mix was vigorously stirred for 90 min, after which 381 mg monochloroacetic acid were added. The reaction was stirred for further 4 h at 65uC in a Heidolph MR 3001 K thermocouple (ß Heidolph Instruments GmbH & Co. KG). The product was recovered by filtration on Whatman # 1, and washed successively with methanol-acetic acid (7:3 v/v), methanol-water (4:1 v/v), methanol and a final step with acetone. The supernatant was filtered again and washed with acetone, allowed to dry overnight, followed by suspension in 150 ml of distilled water, dialyzed overnight against water and finally lyophilized to complete dryness.

Infrared (IR) Spectroscopy
Samples were prepared as KBr pellets. IR spectra were recorded from 3500 to 500 cm 21 , using a Nicolet iS10 IR spectrometer (Thermo Fisher Scientific, Waltham, MA, EE.UU), coupled to the OMNIC 8.0 software.

Enzymatic Assays
All reactions were carried out with 100 mg of Agn1p-his and 1 mg/ml of SCMG in CH 3 COONa buffer (50 mM, 1 h) in a final volume of 1 ml. Reactions were stopped by heating at 100uC [40]. Free reducing ends were analyzed using the colorimetric bicinchoninic acid (BCA) assay [41]. Optimum pH and optimum temperature were determined by performing the reaction at pH values between 4.0 and 7.2, and a temperature range between 23 to 50uC, respectively.
The enzymatic reactions were concentrated to 50 ml by lyophilization. Aliquots of 5 ml were placed on a TLC plate (EMD, 5715-7, TLC Silica Gel 60 F254 20x20 cm), using propanol/water (7/3, v/v) as the mobile phase, for three hours in a closed chamber previously saturated with the solvent mixture. As standard markers, glucose (Himedia, RM 016-500G), maltose Figure 6. Complementation of S. pombe agn1D with the P. brasiliensis AGN1 gene. S. pombe ags1D was complemented with pHV3, which contains the complete P. brasiliensis AGN1 gene, including its original signal peptide coding region (S. pombe strain HLVSP3) (D1, D2) or pHV4, which includes a chimeric P. brasiliensis AGN1, whose signal peptide coding region was substituted by the S. pombe agn1 signal peptide coding region (S. pombe strain HLVSP4) (C1, C3). In both cases, the plasmids restored the wild type phenotype. As positive control, plasmid pREP3X-agn1 + , which includes the complete ORF from the S. pombe agn1 + gene, was transformed into S. pombe ags1D (HLVSP5) (B1 and B2). Negative control consisted of S. pombe ags1D transformed with the empty vector pREP3X (HLVSP6) (A1, A2). White arrows point to the defect in separation at the tip of the daughter cells. Left panel show cells stained with calcofluor white (A1, B1, C1, and D1). Bar  (Sigma, M5885), maltotriose (Sigma, 851,493) at a concentration of 333 mg/ml were placed, in separate contiguous lanes. After completion of the run, the plates were dried at 60uC for 10 min, and impregnated with iodine vapors or a specific staining solution for carbohydrates (KMnO 4 1.5 g; K 2 CO 3 10 g, 1.25 ml NaOH 10% in 200 ml of distilled water) using the spray-type sprayer Flask Aldrich (Z190373 -1EA). Plates were left to dry and developed for about 1 h at 60uC, producing yellow spots on a pink background [43]. The Rf for each lane was calculated by the ratio of the distances traveled by the spots to the distance reached by the front.

S. Pombe Complementation Assay
Two constructions were prepared for use in complementation tests: The first one was obtained by cloning the P. brasiliensis AGN1 ORF into the XhoI and BamHI restriction sites of the S. pombe expression vector pREP3X [44,45], generating plasmid pHV3. The second one, was achieved by replacing the P. brasiliensis AGN1 signal peptide coding sequence from its ORF, with the S. pombe agn1 signal peptide coding sequence, by means of PCR overlap extension [46], using Advantage H 2 DNA Polymerase Mix (Clontech Laboratories, Inc.) and cloning the resulting chimera into the XhoI and BamHI restriction sites of expression vector pREP3X, to produce plasmid pHV4. Oligonucleotide sequences used for amplification of both products can be found in Table 1.
Both plasmids were used to transform S. pombe 1252 (agn1 mutant strain) as described in Suga and Hatakeyama [47], and transformants selected on EMM plates without leucine. As controls, pREP3X::agn1 (expression vector containing the S. pombe agn1 gene, positive control) and pREP3X (empty vector, negative control) were used. Transformants were evaluated by PCR using the primers listed in Table 1.
Complementation of S. pombe 1252 by the P. brasiliensis AGN1 gene was followed by observation of calcofluor white stained cells in a fluorescence microscope Leica DM2000 equipped with H3 filter. Photographs of fluorescent images were taken with a Leica DFC310 FX digital camera, using an immersion objective with 100X magnification. For microscopic observation, 50 ml of cell suspension was mixed with 50 ml of 1 mg/ml calcofluor white (Sigma, F3543). The mixture was smeared onto slides plates pretreated with 20 ml of 0.1% polylysine, air-dried, and washed with PBS. To quantify the degree of complementation, sedimentation assays were performed as in [15].

P. brasiliensis AGN1 Sequence and in Silico Analysis
The P. brasiliensis AGN1 gene has three exons that account for a putative coding region of 1495 bp, separated by two introns, all confirmed by comparison of the sequence of the RT-PCR product with the corresponding genomic sequence ( Figure S1). It encodes a predicted protein of 456 amino acids ( Figure S1), with high identity to fungal glucanases belonging to the glycoside hydrolase family 71 (GH-71) (Neosartorya fischeri 77%, A. fumigatus 76%, A. niger 76%, A. nidulans 74%).
In silico analysis of the deduced protein shows a signal peptide corresponding to the 21 initial amino acids, and a main domain homologous to the GH-71 family, which extends from residues 23 to 432 ( Figure S1), similar to glucanases from S. pombe and A. nidulans [15,16,18]. It presents an estimated mass of 51.2 kDa, and an isoelectric point of 7.1. Also, putative sites for post-translational modifications are present. A hydropathic profile plot shows that the Agn1p sequence is predominantly hydrophilic except for three slightly hydrophobic areas, with no transmembrane domains (not shown).
A search in the P. brasiliensis genome database (http://www. broadinstitute.org/) shows that AGN1 is the only gene in the P. brasiliensis genome related to the hydrolysis of a-1,3-glucan. A Clustal analysis was performed that included 90 complete amino acid sequences of fungal glucanases present in GenBank and CAZy databases (http://www.cazy.org/GH71_eukaryota.html), grouping P. brasiliensis Agn1p into the glycoside hydrolase family 71 (Table 2, Figure 1). Variations among amino acid sequences allow us to propose a subdivision in the family 71 of glycoside hydrolases into 5 sub-groups (G1, G2, G3, G4 and G5; Figure 1).

AGN1 Transcription Analysis under Horse Serum Supplementation
Supplementation of growth medium with 5% horse serum (HS) has been reported as a booster for a-1,3-glucan synthesis in P. brasiliensis [12]. A qPCR expression analysis of P. brasiliensis AGN1 and AGS1 (encoding for a-1,3-glucan synthase, [12]) showed that their transcriptional levels were sharply increased in the presence of HS (Figure 2A and 2B), which agrees with the reported increase in cell wall a-1,3-glucan under supplementation of the culture medium with HS [12].

Agn1p Heterologous Expression, and Biochemical Characterization
Protein expression of P. brasiliensis a-glucanase (Agn1p) was performed, using E. coli as the expression host. The cDNA without the signal peptide coding sequence was cloned into the pQE30Xa plasmid in frame with the His-tag present in the commercial plasmid, to produce the pHV2 vector. Induction of protein expression was obtained by addition of IPTG and the protein purified by affinity chromatography. A SDS-PAGE of the purified Agn1p-His showed a single band with an estimated molecular mass of 51.8 kDa (R 2 = ,0.98; Figure 3A and 3B), in close agreement with its calculated molecular mass of 51.2 kDa. A western analysis using antibody directed to the RGS-His epitope, confirmed that the band corresponds to the purified protein fused to the histidine tag ( Figure 3B). A lower molecular weight band can also be seen, which may correspond to the degradation of Agn1p at the C-terminus, because the recorded signal shows the presence of RGS-His epitope located at the N-terminal region.
Agn1p was only active against a-(1,3)-glucan (SCMG) when tested against a battery of glucose or glucosamine polymers (laminarin, starch, cellulose, chitin and dextran) ( Figure 4B). Optimal reaction conditions for P. brasiliensis Agn1p were established at 1 h as pH 5.0 and 40uC (not shown). No inhibitory effect was observed upon Agn1p-his pre-incubation with inhibitors of exo-catalytic hydrolases (1-deoxynojirimycin and D-glucono-1,5-lactone) ( Figure 4A). Endo-catalytic activity of AGN1 was determined by TLC analysis (Figure 5), where heptasaccharides (R 2 = ,0.9786) were the main hydrolysis products. AGN1 from P. brasiliensis Complements the Septation Phenotype of S. pombe agn1D Mutant For complementation, two different plasmids were introduced into S. pombe agn1 null mutant strain 1252: (a) pHV3, containing the complete P. brasiliensis AGN1 gene, including its original signal peptide coding region (S. pombe strain HLVSP3), and (b) pHV4, which includes a chimeric P. brasiliensis AGN1, whose signal peptide coding region was substituted by the S. pombe agn1 signal peptide coding region, constructed by PCR overlap extension (S. pombe strain HLVSP4). As positive control, S. pombe HLVSP5, containing plasmid pREP3X-agn1 + , which includes the complete ORF from the S. pombe agn1 + gene, was used. Negative control consisted of S. pombe 1252 transformed with the empty vector pREP3X, (HLVSP6 strain). Cells were analyzed by calcofluor white staining, confirming that the strains carrying the agn1 + and AGN1 ORFs were able to suppress the separation defect shown by the S. pombe 1252 mutant (Figure 6), a result confirmed by sedimentation assays (Table 3).

Discussion
P. brasiliensis strain Pb-73 has a single a-1,3-glucanase-encoding gene (AGN1) interrupted by two introns (accession number EF679780), whose product, Agn1p, is 77% identical to other fungal a-1,3-glucanases. P. brasiliensis Agn1p has a molecular mass of ,51 kDa after SDS/PAGE; according to its amino acid sequence it can be classified into the poorly characterized family 71 of glycoside hydrolases (GH-71). A clustal alignment of P. brasiliensis Agn1p with other fungal GH-71 allows us to infer the location of 5 conserved residues, specifically aspartic and glutamic acids (D and E respectively), which may correspond to amino acids involved in the acid-base catalytic mechanism [50,51]. Figure 1 shows a phylogram of relationships between different fungal GH-71. Five clearly differentiated clusters can be observed, which allow us to propose a subdivision of fungal GH-71 into at least five groups (G1 to G5) ( Figure 1, Table 2), with P. brasiliensis Agn1p clustering into G1. G3, G4 and G5 share the conserved sequence (T/S)WND, while G4 and G2 shared the consensus sequence: FALN. It should be noted that glucanases that exhibit a mutan-binding domain (MBD) are grouped exclusively within the group G4 (Hypocrea lixii, Trichoderma asperellum, Penicillium purpurogenum), showing high identity to T. harzianum MBD (from 51 to 86%, [59]. Every group also presents specific conserved signatures: The first 20 amino acid of P. brasiliensis Agn1p are predicted to be a signal peptide, suggesting the location of Agn1p towards the P. brasiliensis membrane or cell wall, where the a-1,3-glucan, a virulence factor and the specific substrate for Agn1p, is located. This location is shared by most of the fungal a-1,3-glucanases so far studied [15,17,13,16,18]. In agreement with the presence of the signal peptide, computationally predicted post-translational modifications were found ( Figure S1). Among them, a sequence for cellular adhesion, described in P. brasiliensis, H. capsulatum, A. fumigatus, C. immitis, for proteins that bind to the extracellular matrix [52,53,54,55,56], and an N-glycosylation site, reported to play a role in post-translational modification of Candida albicans cell wall proteins involved in cell adhesion processes. Despite those possible post-translational modifications, we were able to achieve the purification of functional Agn1p from heterologous expression in E. coli, showing that in the absence of post-translational modifications (due to intracellular heterologous expression) the glucanase activity remains, as was recently reported for a recombinant glucanase from T. harzianum expressed also in E. coli [57]. Such glucanase has a specific activity of 0.097 U/mg, while the P. brasiliensis a-1,3-glucanase, measured at optimal conditions with SCMG as the soluble substrate, had a specific activity of 0.075 U/mg. It should be noted that the conditions used for carboxymethylation have been described as adequate to ensure solubility without alteration of the linear structure of the polysaccharide [39]. IR and 13 C-NMR ( Figure S3) spectra of SCMG indicate that carbonyl groups were properly added to the otherwise unchanged polysaccharide, data that support the effectiveness of the reaction, and the maintenance of an a anomeric configuration in the resulting SCMG [49]. P. brasiliensis Agn1p enzyme showed high specificity for its proposed natural substrate, cell wall a-1,3-glucan (SCMG, Figure 4B). The enzyme had an endo-catalytic activity, as deduced from TLC results ( Figure 5, oligosaccharides as reaction products) and the lack of inhibitory effects by exo-catalytic inhibitors of hydrolases ( Figure 4A). This high specificity and cutting pattern is shared with S. pombe, P. purpurogenum and T. harzianum glucanases [17].
Gene expression analyses by real-time PCR for both AGN1 and AGS1 in the Y phase (Fig. 2), showed significant increases (2 to 2.5 times transcript levels) in the expression of both genes when growing the pathogenic Y phase in the presence of horse serum, which boosts the synthesis of cell wall a-1,3-glucan, as previously reported [12]. This result suggests that the increased expression of AGN1 in P. brasiliensis is related to an increase in cell wall a-1,3glucan in the Y phase of this fungus.
Functionality of the P. brasiliensis AGN1 gene was demonstrated by complementation of S. pombe strain 1252, an agn1 null mutant. This mutation produces cell clumps due to the inability of motherdaughter cells to split, once the glucanase required for the hydrolysis of the septal a-1,3-glucan is unavailable. In S. pombe, the septum is formed by a primary septum (mainly b-1,3-glucan), surrounded by a secondary septum (a mixed structure of a-1,3glucan, 1,6-branched 1,3-b-glucan, 1,6-b-glucan and galactomannans), through which septum degradation and cell separation starts. Therefore, agn1 mutants are incapable of splitting, as shown with calcofluor white staining ( Figure 6, A1-A2) [58]. The separation of the two daughter cells in S. pombe is initiated through secondary septum degradation; hence, the absence of a-1,3glucanase activity prevents the splitting of the primary septum. Expression of P. brasiliensis AGN1 into S. pombe agn1D, either with its original signal peptide-coding region or as a chimera with the P. brasiliensis signal peptide-coding region substituted by S. pombe agn1 signal peptide-coding region, restored the wild type phenotype (Figure 6, C1,C2 and D1, D2; Table 3), and demonstrated the functionality of P. brasiliensis AGN1. This fact, plus the high specificity shown by P. brasiliensis a-1,3-endoglucanase, suggest the involvement of this enzyme in the yeast phase cytokinesis.
The fact that P. brasiliensis genome presents a single AGN1 gene seems to be in consonance with the presence of a single a-1,3glucan synthase (AGS1) gene recently reported [12]. Ags1p is associated with the synthesis of cell wall a-1,3-glucan, a proposed virulence factor in P. brasiliensis, and found to contribute to pathogenesis in H. capsulatum by concealing immunostimulatory bglucans from detection by host phagocytic cells [5,7]. Unlike the metabolism of chitin, which depends on up to seven different chitin synthases [59, 60. 61], and several chitinases [62,63], the apparent simplicity of the mechanisms of synthesis and hydrolysis of P. brasiliensis a-1,3-glucan (one synthase, one hydrolase), and the fact that this polysaccharide is absent from the natural fungal host, leads us to propose both, its mechanisms of synthesis (by blocking it) and degradation (by stimulating it) as potential targets for the development of specific drugs against P. brasiliensis, which might result in the depression of fungal virulence, and allow the action of the natural immune response of the infected organism against the fungus. Figure S1 AGN1 genomic sequence (gDNA) from P. brasiliensis strain Pb-73. Highlighted in yellow, the deduced amino acid sequence of P. brasiliensis a-1,3-glucanase Agn1p. Highlighted in italics and bold, the putative start codon and the methionine residue attached, respectively. In red letters, 21 amino acids belonging to a putative signal peptide. In green, AGN1 intron sequences, (their processing sites are underlined). Post-translational putative modification sites are highlighted in colored boxes: blue: cell adhesion; purple, N-glycosylation. (TIF) Figure S2 SCMG 13 C-NMR spectra. The box indicates the location of the signals corresponding to the carbonyl group, while the arrows point to the signature band of the a-1,3 configuration of both SCMG and a-1,3-glucan. (TIF)