Antifungal activities of wood extractives

Extractives are non-structural wood molecules that represent a minor fraction in wood.However, they are source of diverse molecules putatively bioactive. Inhibition of fungal growth is one of the most interesting properties of wood extractives in a context of wood preservation, crop protection or medical treatments.The antifungal effect of molecules isolated from wood extractives has been mainly attributed to various mechanisms such as metal and free radical scavenging activity, direct interaction with enzymes, disruption of membrane integrity and perturbation of ionic homeostasis. Lignolytic fungi, which are microorganisms adapted to wood substrates, have developed various strategies to protect themselves against this toxicity.A better knowledge of these strategies could help both developing new systems for extractive removal in biomass valorization processes and using these molecules as antifungal agents. (C) 2017 British Mycological Society

Evolutionary divergence of Ure2pA glutathione transferases in wood degrading fungi

International audienceThe intracellular systems of detoxification are crucial for the survival of wood degrading fungi. Within these systems, glutathione transferases could play a major role since this family of enzymes is specifically extended in lignolytic fungi. In particular the Ure2p class represents one third of the total GST number in Phanerochaete chrysosporium. These proteins have been phylogenetically split into two subclasses called Ure2pA and Ure2pB. Ure2pB can be classified as Nu GSTs because of shared structural and functional features with previously characterized bacterial isoforms. Ure2pA can rather be qualified as Nu-like GSTs since they exhibit a number of differences. Ure2pA possess a classical transferase activity, a more divergent catalytic site and a higher structural flexibility for some of them, compared to Nu GSTs. The characterization of four members of this Ure2pA subclass (PcUre2pA4, PcUre2pA5, PcUre2pA6 and PcUre2pA8) revealed specific functional and structural features, suggesting that these enzymes have rapidly evolved and differentiated, probably to adapt to the complex chemical environment associated with wood decomposition

Data from: Characterization of glutathione transferases involved in the pathogenicity of Alternaria brassicicola

Background: Glutathione transferases (GSTs) represent an extended family of multifunctional proteins involved in detoxification processes and tolerance to oxidative stress. We thus anticipated that some GSTs could play an essential role in the protection of fungal necrotrophs against plant-derived toxic metabolites and reactive oxygen species that accumulate at the host-pathogen interface during infection. Results: Mining the genome of the necrotrophic Brassica pathogen Alternaria brassicicola for glutathione transferase revealed 23 sequences, 17 of which could be clustered into the main classes previously defined for fungal GSTs and six were ‘orphans’. Five isothiocyanate-inducible GSTs from five different classes were more thoroughly investigated. Analysis of their catalytic properties revealed that two GSTs, belonging to the GSTFuA and GTT1 classes, exhibited GSH transferase activity with isothiocyanates (ITC) and peroxidase activity with cumene hydroperoxide, respectively. Mutant deficient for these two GSTs were however neither more susceptible to ITC nor less aggressive than the wild-type parental strain. By contrast mutants deficient for two other GSTs, belonging to the Ure2pB and GSTO classes, were distinguished by their hyper-susceptibility to ITC and low aggressiveness against Brassica oleracea. In particular AbGSTO1 could participate in cell tolerance to ITC due to its glutathione-dependent thioltransferase activity. The fifth ITC-inducible GST belonged to the MAPEG class and although it was not possible to produce the soluble active form of this protein in a bacterial expression system, the corresponding deficient mutant failed to develop normal symptoms on host plant tissues. Conclusions: Among the five ITC-inducible GSTs analyzed in this study, three were found essential for full aggressiveness of A. brassicicola on host plant. This, to our knowledge is the first evidence that GSTs might be essential virulence factors for fungal necrotrophs

The structure of Trametes versicolor glutathione transferase Omega 3S bound to its conjugation product glutathionyl-phenethylthiocarbamate reveals plasticity of its active site

Trametes versicolor glutathione transferase Omega 3S (TvGSTO3S) catalyzes the conjugation of isothiocyanates (ITC) with glutathione (GSH).Previously, this isoform was investigated in depth both biochemically and structurally. Structural analysis of complexes revealed the presence of a GSH binding site (G site) and a deep hydrophobic binding site (H site) able to bind plant polyphenols. In the present study, crystals of apo TvGSTO3S were soaked with glutathionyl-phenethylthiocarbamate, the product of the reaction between GSH and phenethyl isothiocyanate (PEITC).On the basis of this crystal structure, we show that the phenethyl moiety binds in a new site at loop beta(2)-alpha(2) while the glutathionyl part exhibits a particular conformation that occupies both the G site and the entrance to the H site. This binding mode is allowed by a conformational change of the loop beta(2)-alpha(2) at the enzyme active site. It forms a hydrophobic slit that stabilizes the phenethyl group at a distinct site from the previously described H site.Structural comparison of TvGSTO3S with drosophila DmGSTD2 suggests that this flexible loop could be the region that binds PEITC for both isoforms. These structural features are discussed in a catalytic context

Interactions between fungal glutathione transferases and wood extractives

International audienceThe interactions between extractives from several tree species of French Guyana and glutathione transferases (GST) from the white-rot Trametes versicolor have been studied. These interactions have been monitored using a thermal shift assay. It appears from these experiments that TvGSTO2S interacts strongly with acetonic extract of Bagassa guianensis and in particular with oxyresveratrol. In agreement with previous results, the obtained data suggest that the GST network could give insights on the adaptation of wood decaying fungi to their chemical environment

Oak extractive-induced stress reveals the involvement of new enzymes in the early detoxification response of Phanerochaete chrysosporium

International audienceExtensive evidence showed that the efficiency of fungal wood degradation is closely dependent on their ability to cope with the myriad of putative toxic compounds called extractives released during this process. By analysing global gene expression of Phanerochaete chrysosporium after short oak extractive treatment (1, 3 and 6 h), we show that the early molecular response of the fungus concerns first mitochondrial stress rescue followed by the oxidation and finally conjugation of the compounds. During these early responses, the lignolytic degradative system is not induced, rather some small secreted proteins could play an important role in cell protection or signaling. By focusing on the functional characterization of an hitherto uncharacterized glutathione transferase, we show that this enzyme interacts with wood molecules suggesting that it could be involved in the detoxification of some of them, or act as a scavenger to prevent their cytosolic toxicity and favour their transport

Glutathione transferases and wood extracts

International audienceWood rotting fungi have developed an efficient detoxification system allowing them to be well adapted to their way of life, these fungi generating and encountering potential toxic compounds during wood degradation. Belonging to this detoxification network, glutathione transferases are particularly extended in these fungi and their presence could reflect the chemical environments encountered by the fungi. To test this hypothesis, we developed biochemical assays to quantify interactions between wood extracts and glutathione transferases from from two white-rot fungi: Trametes versicolor and Phanerochaete chrysosporium. The obtained preliminary results reported in this paper demonstrate that GSTs from white-rot fungi could be used to discriminate wood extracts from different origins in accordance with their chemical compositions

Glutathione Transferases: Surrogate Targets for Discovering Biologically Active Compounds

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Emerging function for a glutathione transferase in response to wood extractives toxicity

International audienceThe first steps of wood degradation by fungi lead to the release of toxic compounds known as extractives. To better understand how lignolytic fungi cope with these products, a transcriptomic analysis of Phanerochaete chrysosporium genes was performed in presence of oak acetonic extractives. The most up-regulated genes relate to nutrition, nucleic acid modification, gene regulation, signalling and stress responses. Focusing on the induced antioxidant and detoxification systems, a glutathione transferase of the GTT2 class has been selected for functional characterization. This enzyme does not possess any classical glutathione transferase activity but rather a peroxidase activity. The occurrence of the GTT2 gene within the genome is closely linked to the wood decay capabilities of the fungi. This example suggests that the intracellular detoxification system could have evolved concomitantly with the extracellular ligninolytic machinery in relation to the capacity to degrade wood

Trametes versicolor glutathione transferase Xi 3, a dual Cys-GST with catalytic specificities of both Xi and Omega classes

Glutathione transferases (GSTs) from the Xi and Omega classes have a catalytic cysteine residue, which gives them reductase activities.Until now, they have been assigned distinct substrates. While Xi GSTs specifically reduce glutathionyl-(hydro)quinones, Omega GSTs are specialized in the reduction of glutathionyl-acetophenones. Here, we present the biochemical and structural analysis of TvGSTX1 and TvGSTX3 isoforms from the wood-degrading fungus Trametes versicolor. TvGSTX1 reduces GS-menadione as expected, while TvGSTX3 reduces both Xi and Omega substrates. An in-depth structural analysis indicates a broader active site for TvGSTX3 due to specific differences in the nature of the residues situated in the C-terminal helix α9.This feature could explain the catalytic duality of TvGSTX3. Based on phylogenetic analysis, we propose that this duality might exist in saprophytic fungi and ascomycetes