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

Abstract

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.

An Amylase-Like Protein, AmyD, Is the Major Negative Regulator for α-Glucan Synthesis in Aspergillus nidulans during the Asexual Life Cycle

“…Therefore, the functions of glucanases are independent from AmyD. Evidence from other α-glucanase characterization work also showed that α-glucanase is functional by itself [ 14 , 15 , 16 ]. In addition, we noticed that, when AgnB or MutA was overexpressed in an AmyDΔ background, the effect from AmyD deletion was abolished (compare data in [ 10 ] with Figure 3 D and Figure 4 C).…”

“…In S. pombe , α-glucanase ( Sp Agn1p) was shown to have an endo-catalytic hydrolysis ability on α-glucan (hydrolyzing α-glucan into pentasaccharides), and was thereby important for successful cell division [ 14 ]. Paracoccidioides brasiliensis α-glucanase ( Pb Agn1p) had comparable activity [ 15 ]. In Trichoderma species, two α-glucanases have been characterized, and these were suggested to have antifungal effects because their expression was highly induced under antagonistic conditions [ 16 , 17 ].…”

Effects of Argentilactone on the Transcriptional Profile, Cell Wall and Oxidative Stress of Paracoccidioides spp.

“…lutzii were decreased when treated with argentilactone. Studies have demonstrated the importance of genes related to cell wall biosynthesis in Paracoccidioides spp., including chitin synthase genes [ 9 , 44 – 48 ].…”

Coordinating septum formation and the actomyosin ring during cytokinesis in Schizosaccharomyces pombe

“…While the authors report that addition of the drug aculeacin A (blocks glucan synthesis) does not lead to ring sliding, Bgs1 is resistant to this drug and thus one cannot conclude if the Bgs1 enzymatic activity is necessary for anchoring the ring (Castro et al, 1995;Martins et al, 2011). Bgs1 has been reported to cooperate with Paxillin Pxl1 and Cdc15 to maintain ring integrity and to Martin-Cuadrado et al (2003), Wang et al (2002), Garcia et al (2005), Sipiczki et al (2014), Martin-Cuadrado et al (2008), Wang et al (2003;2015), Perez et al (2015), Santos et al (2005), Munoz et al (2014), Villalobos-Duno et al (2013), Nakano et al (2003) promote septum synthesis (Cortes et al, 2015). Similar to cdc15 and bgs1 mutants, pxl1 mutants also show ring sliding defects (Cortes et al, 2015).…”