Glutathione reductase/glutathione is responsible for cytotoxic elemental sulfur tolerance via polysulfide shuttle in fungi

Abstract

Fungi that can reduce elemental sulfur to sulfide are widely distributed, but the mechanism and physiological significance of the reaction have been poorly characterized. Here, we purified elemental sulfur-reductase (SR) and cloned its gene from the elemental sulfur-reducing fungus Fusarium oxysporum. We found that NADPH-glutathione reductase (GR) reduces elemental sulfur via glutathione as an intermediate. A loss-of-function mutant of the SR/GR gene generated less sulfide from elemental sulfur than the wild-type strain. Its growth was hypersensitive to elemental sulfur, and it accumulated higher levels of oxidized glutathione, indicating that the GR/glutathione system confers tolerance to cytotoxic elemental sulfur by reducing it to less harmful sulfide. The SR/GR reduced polysulfide as efficiently as elemental sulfur, which implies that soluble polysulfide shuttles reducing equivalents to exocellular insoluble elemental sulfur and generates sulfide. The ubiquitous distribution of the GR/glutathione system together with our findings that GR-deficient mutants derived from Saccharomyces cerevisiae and Aspergillus nidulans reduced less sulfur and that their growth was hypersensitive to elemental sulfur indicated a wide distribution of the system among fungi. These results indicate a novel biological function of the GR/glutathione system in elemental sulfur reduction, which is distinguishable from bacterial and archaeal mechanisms of glutathione- independent sulfur reduction.

FIGURE 1.

Elemental sulfur reduction and toxicity. A, sulfide production by F. oxysporum JCM11502 (WT) incubated in MMEA with various amounts of CS (circles) and PS (triangles) for 24 h at 30 °C. B, numbers of surviving cells after similar incubation with various concentrations of CS (circles), PS (triangles), and sodium sulfide (squares). C, morphology of colonies on MMEA agar plates incubated for 48 h with or without CS and sodium sulfide. D, time-dependent change in sizes of colonies on plates with 5 mm CS (circles), 5 mm sodium sulfide (squares), and no additions (triangles). E and F, effects of thiolate reagents. F. oxysporum WT cells were incubated with or without DTNB, IAA, and IAM (5 mm each) for 2 h, and then sulfide produced within 15 min was monitored (E). Intracellular GSH (filled bars) and GSSG (unfilled bars) were measured in these cells (F). *, less than 1.0 nmol-S mg⁻¹.

FIGURE 2.

Sulfur reductase activities of F. oxysporum. A, cell-free extracts were prepared from F. oxysporum WT (W) and DGR (D) strains cultured with 20 mm eq. PS at 30 °C for 24 h, and rates of sulfide production (left) and NADPH oxidation (right) were determined as described under “Experimental Procedures.” Concentrations of electron mediators and acceptors were set at 1 mm (GSSG), 10 μm (TrxA), 5 mm eq. (CS), and 0.1 mm (polysulfide; polyS). Data are means of three experiments, and error bars represent standard errors. *, less than 0.5 nmol min⁻¹ mg⁻¹. B, reaction catalyzed by SR and polySR. C, amounts of glrA gene transcript determined by quantitative PCR in WT cultured with (+S) or without (−S) 20 mm eq. PS at 30 °C for 12 h. D, enzyme activities of SR (filled bars) and GR (unfilled bars) in WT cultured for 24 h under identical conditions.

FIGURE 3.

Sulfur/glutathione reductase is involved in sulfur tolerance. A, strategy for homologous recombination into glrA locus to construct glrA mutants (left) and Southern blot analysis (right) of WT (lane 1) and DGR (lane 2) strains. Total DNA from strains was digested with EcoRI before blotting and hybridization. B, intracellular GSH and GSSG concentrations. WT and DGR were incubated in MMEA with (+S) or without (−S) 20 mm eq. PS for 24 h at 30 °C. Filled and unfilled bars represent GSH and GSSG, respectively. C, time-dependent production of sulfide by WT (closed) and DGR (open). Strains were cultured in MMEA medium containing 5 mm eq. CS (circles) and 20 mm eq. PS (triangles). Culture flasks were sealed with butyl rubber caps to prevent sulfide evaporation. D, morphology of colonies appeared on MMEA agar plates with or without sodium sulfide, CS, and polysulfide (polyS) (1 mm each) after incubation for 30 h.

FIGURE 4.

Role of GR in oxidative stress responses. A, effects of menadione (MD), diamide (DA), and hydrogen peroxide (H₂O₂) (1 mm each) on growth of WT and DGR. Strains were incubated on MMEA for 72 h at 30 °C. B, intracellular activities of thioredoxin reductase (TRR), catalase, and cytochrome c peroxidase (CPX). WT and DGR were incubated in MMEA with (+S) or without (−S) 20 mm eq. PS for 24 h at 30 °C.

FIGURE 5.

Sulfur reduction mediated by SR/GR in F. oxysporum and other fungi. A, model of SR/GR-mediated sulfur reduction. B, sulfide production by WT (filled bars) and GR gene disruptants (unfilled bars) of S. cerevisiae and A. nidulans. S. cerevisiae strains BY4741 (WT) and glr1Δ (Δglr1) were used to inoculate medium containing either 20 mm eq. PS or 5 mm eq. CS to optical density of 0.4. The A. nidulans WT and DGR1 (ΔglrA) strains (20) (100 mg dry cells) were cultured in MMEA medium containing 20 mm eq. PS or 5 mm eq. CS for 24 h. C, morphology of S. cerevisiae colonies on MMEA agar plates with or without sodium sulfide, CS, and polysulfide (polyS) (1 mm each) after incubation for 38 h. D, morphology of A. nidulans colonies on MMEA agar plates with or without sodium sulfide, CS, and polysulfide (polyS) (1 mm each) after incubation for 48 h.