The glutathione system of Aspergillus nidulans involves a fungus-specific glutathione S-transferase

Abstract

The tripeptide glutathione is involved in cellular defense mechanisms for xenobiotics and reactive oxygen species. This study investigated glutathione-dependent mechanisms in the model organism Aspergillus nidulans. A recombinant dimeric protein of A. nidulans glutathione reductase (GR) contained FAD and reduced oxidized glutathione (GSSG) using NADPH as an electron donor. A deletion strain of the GR gene (glrA) accumulated less intracellular reduced glutathione (GSH), indicating that the fungal GR contributes to GSSG reduction in vivo. Growth of the deletion strain of glrA was temperature-sensitive, and this phenotype was suppressed by adding GSH to the medium. The strain subsequently accumulated more intracellular superoxide, and cell-free respiration activity was partly defective. Growth of the strain decreased in the presence of oxidants, which induced glrA expression 1.5-6-fold. These results indicated that the fungal glutathione system functions as an antioxidant mechanism in A. nidulans. Our findings further revealed an initial proteomic differential display on GR-depleted and wild type strains. Up-regulation of thioredoxin reductase, peroxiredoxins, catalases, and cytochrome c peroxidase in the glrA-deletion strain revealed interplay between the glutathione system and both the thioredoxin system and hydrogen peroxide defense mechanisms. We also identified a hypothetical, up-regulated protein in the GR-depleted strains as glutathione S-transferase, which is unique among Ascomycetes fungi.

FIGURE 1.

Localization of GlrA-GFP fusion in A. nidulans. Conidia of transformants harboring pGRgfp1 (A and B) or pGRgfp1-M86A (C and D) that, respectively, produce GlrA-GFP and GlrA^M86A-GFP, were incubated in MMDN medium at 37 °C for 10 h. GlrA-GFP fusion proteins (A and C) and mitochondria stained with MitoTracker Red CMXRos (B and D) were observed by fluorescence microscopy. Scale bars, 10 μm.

FIGURE 2.

Construction of glrA mutant of A. nidulans. A, strategy for homologous recombination into glrA locus to construct glrA deletion mutants. kb, kilobases. B, Southern blot analysis of A. nidulans WT (FGSC A26) (lane 1) and DGR1 (lane 2). Total DNA from strains was digested with PstI before blotting and hybridization. C, strains were grown on MMDN agar plates with or without 10 mm GSH at indicated temperatures for 72 h. Radii of colonies are indicated in mm with S.E. D, conidia of WT (circles) and DGR1 (squares) strains of A. nidulans were spread onto MMDN agar plates with (open) or without (closed) 10 mm GSH and incubated at 30 °C for 120 h. Relative germination rates were determined by setting number of colonies on MMDN agar plates (without oxidant) as 100%. Data are the means of three experiments. Error bars represent S.E. E, mycelia of WT and DGR1 strains of A. nidulans were incubated for 6 h at 30 °C, and intracellular glutathione was determined. Closed and open bars represent reduced and oxidized glutathione, respectively. S.E. are <20%.

FIGURE 3.

Effects of glrA mutation on cellular processes. A, intracellular levels of superoxide and H₂O₂ in WT (closed bars) and DGR1 (open bars) strains of A. nidulans cultured at 30 °C for 20 h. Data are means of four experiments. Error bars represent S.E. p < 0.03 (superoxide) and p < 0.05 (H₂O₂). B, respiration activity of WT (closed bars) and DGR1 (open bars) strains of A. nidulans cultured at 30 °C for 6 h after pre-cultivating conidia at 30 °C for 20 h. Data are the means of four experiments. Error bars represent S.E. C, aconitase (left) and sulfite reductase (right) activity of WT (closed bars) and DGR1 (open bars) cultured at 30 °C for 6 h after pre-cultivating conidia at 30 °C for 20 h. Data are the means of four experiments. Error bars represent S.E.

FIGURE 4.

Effects of oxidative stress on A. nidulans DGR1. A, quantitative PCR analysis of glrA gene transcript. A. nidulans was incubated at 30 °C for 6 h, the indicated compounds were added or temperatures were shifted, and then incubation proceeded for further 3 h. Bars indicate values reported as relative expression rates obtained by RT-PCR and normalized to actA. B, strains were grown on MMDN agar plates with or without various oxidants at 30 °C for 120 h. Concentrations of oxidants: 30 μm (menadione (MD)), 2 mm (diamide), and 2.5 mm (hydrogen peroxide). Radii of colonies are indicated in mm with S.E. C-E, conidia of WT (circles) and DGR1 (squares) strains of A. nidulans were spread onto MMDN agar plates and incubated at 30 °C for 120 h. Relative germination rates were determined by setting number of colonies on MMDN agar plates (without oxidant) as 100%. Data are the means of three experiments. Error bars represent S.E.

FIGURE 5.

Characterization of novel GST from A. nidulans. A, SDS-PAGE of purified enzyme. Purified enzymes (1 μg) were resolved by SDS-PAGE on 10% polyacrylamide gels and stained with Coomassie Brilliant Blue. Lane 1, markers (Novex Sharp Protein Standard kit, Invitrogen); lane 2, rGstB. B, Quantitative PCR analysis of gstB gene transcript. A. nidulans A26 was grown at 30 °C for 20 h, the indicated compounds were added or temperatures were shifted, and then incubation proceeded for further 3 h. Bars indicate values reported as relative expression rates obtained by RT-PCR and normalized to 18 S rRNA. C, phylogenetic relationships among GstB-like GSTs from various organisms. Multiple alignment and neighbor joining were performed using Molecular Evolutionary Genetics Analysis (MEGA) Software Version 4.0. Accession numbers are from Broad Institute. GstA involved in cluster 2 GST from A. nidulans served as outgroup.