Cell walls of the dimorphic fungal pathogens Sporothrix schenckii and Sporothrix brasiliensis exhibit bilaminate structures and sloughing of extensive and intact layers

This work was supported by the Fundação Carlos Chagas de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ), grants E-26/202.974/2015 and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), grants 229755/2013-5, Brazil. LMLB is a senior research fellow of CNPq and Faperj. NG acknowledged support from the Wellcome Trust (Trust (097377, 101873, 200208) and MRC Centre for Medical Mycology (MR/N006364/1). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Peer reviewedPublisher PD

Biochemical characterization of Paracoccidioides brasiliensis α-1,3-glucanase agn1p, and its functionality by heterologous expression in Schizosaccharomyces pombe

This is an open-access article distributed under the terms of the Creative Commons Attribution License.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.This work was partially supported by Project ICGEB-VEN05 of the International Centre for Genetic Engineering and Biotechnology, Trieste, Italy. HV-D was recipient of a Ph.D scholarship awarded by FONACIT (Fondo Nacional de Ciencia, Tecnología e Innovación) Caracas, Venezuela. MP was recipiente of a MSc scholarship awarded by IVIC, Caracas Venezuela.Peer Reviewe

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

Sedimentation analysis of <i>S. pombe</i> strain 1252 (<i>agn1</i>Δ), complemented with <i>AGN1</i> from <i>P. brasiliensis</i>.

<p>The values presented are the mean ± SD of four individual experiments.</p

Complementation of <i>S. pombe agn1</i>Δ with the <i>P. brasiliensis AGN1</i> gene.

<p><i>S. pombe ags1</i>Δ was complemented with pHV3, which contains the complete <i>P. brasiliensis AGN1</i> gene, including its original signal peptide coding region (<i>S. pombe</i> strain HLVSP3) (D1, D2) or pHV4, which includes a chimeric <i>P. brasiliensis AGN1</i>, whose signal peptide coding region was substituted by the <i>S. pombe agn1</i> signal peptide coding region (<i>S. pombe</i> strain HLVSP4) (C1, C3). In both cases, the plasmids restored the wild type phenotype. As positive control, plasmid pREP3X-<i>agn1⁺</i>, which includes the complete ORF from the <i>S. pombe agn1⁺</i> gene, was transformed into <i>S. pombe ags1</i>Δ (HLVSP5) (B1 and B2). Negative control consisted of <i>S. pombe ags1</i>Δ 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 20 µm.</p

Oligonucleotides used in the amplification of PCR products for the complementation of <i>S. pombe.</i>

1<p>Annealing temperature 53°C.</p>2<p>Annealing temperature for overlap extension reactions 55°C (AB, CD and AD).</p

Sequences used for alignments and phylogenetic tree construction.

*<p>Obtained in this work.</p><p>∼From <i>Paracoccidioides brasiliensis</i> sequence data bank: <a href="http://www.broadinstitute.org/annotation/genome/paracoccidioides_brasiliensis/MultiHome.html" target="_blank">http://www.broadinstitute.org/annotation/genome/paracoccidioides_brasiliensis/MultiHome.html</a>.</p

<i>P.</i><i>brasiliensis</i> Agn1p is a specific endo α-1,3-glucanase.

<p>(<b>A</b>) Inhibition profile of exo-glucoamylase from <i>A. niger</i> (gray) and endo-α-1,3-glucanase from <i>P. brasiliensis</i> (black). Note that none of the indicated inhibitors reduced Agn1p-his activity significantly, even at a high concentration of 250 µM. (<b>B</b>) Agn1p substrate specificity. Purified Agn1p-his was incubated with the indicated substrates at 1 mg/ml. Reactions were carried out at optimal conditions by triplicate.</p

SDS-PAGE, and Western analysis of <i>P.</i><i>brasiliensis</i> Agn1p.

<p>Ni-NTA-purified Agn1p from cell lysates of <i>E. coli</i> transformed with of pQE-30Xa::<i>AGN1</i> (Agn1p), and with the empty pQE-30Xa expression vector as negative control (NC) were separated by SDS-PAGE and stained with coomasie blue (A). The Ni-NTA-purified lysates were blotted on a nitrocellulose membrane and the His-tagged <i>P. brasiliensis</i> Agn1p (Agn1p) visualized using an anti RGS-His antibody (B). E stands for eluate, and NB for unbound material. MW: molecular weight marker. 6HP: 6xHis Ladder. Black arrow signals Agn1p position in both panels.</p

Thin Layer Chromatography (TLC).

<p><i>P. brasiliensis</i> Agn1p was incubated for 1 h with CMGS. Lanes: 1, glucose (G1); 2, maltose (G2); 3, maltotriose (G3); 4, Agn1p incubated with GMGS; 5, CMGS.</p