Surface modified sulfur nanoparticles can escape the glutathione reductase mediated detoxification system in fungi

The antifungal effects of orthorhombic (~10 nm; spherical) and monoclinic (~50 nm; tetrapod) sulfur nanoparticles (SNPs) were studied against the NADPH-dependent glutathione reductase (GR) mediated xenobiotic detoxification system (GSH-GSSG) in filamentous fungi (Aspergillus niger as a model organism). Both the SNPs induced significant reduction in fungal growth and spore formation, and also introduced marked deformities on the surface of conidiophores at their sub-inhibitory concentrations. A genome wide transcriptome profile then revealed manifold reduction in the expression of GSH-GSSG transcripts among SNPs treated fungal isolates, which is unusual for the micron sized elemental sulfur but probably effective in terms of antifungal efficacy.

💡 Research Summary

This study investigates how surface‑modified sulfur nanoparticles (SNPs) circumvent the glutathione reductase (GR)‑mediated detoxification system in the filamentous fungus Aspergillus niger. Two allotropes of sulfur were prepared: orthorhombic (α‑SNP) particles of roughly 10 nm diameter, rendered spherical by polyethylene glycol (PEG) coating, and monoclinic (β‑SNP) tetrapod particles of about 50 nm, stabilized with a Span‑80/Tween‑80 polymer shell. High‑resolution transmission electron microscopy confirmed the size and morphology of each formulation.

Antifungal activity was assessed using a modified agar dilution method (ADM). The minimal inhibitory concentrations (MICs) were 8000 ppm for α‑SNP and 3200 ppm for β‑SNP. Even at sub‑inhibitory concentrations (SICs), both nanoparticle types caused a dose‑dependent reduction in radial colony growth, spore production, and, notably, severe surface deformities of conidiophores as visualized by field‑emission scanning electron microscopy. In contrast, bulk elemental sulfur (ES) showed negligible activity under the same conditions.

To explore the molecular basis of this heightened toxicity, whole‑genome microarray analysis was performed on untreated, ES‑treated, and SNP‑treated cultures. ES exposure up‑regulated key genes of the GSH‑GSSG pathway—including glutathione reductase (GR), γ‑glutamylcysteine synthetase (GCS), and glutathione synthetase (GS)—by 8–11‑fold, reflecting an active detox response. By comparison, α‑SNP treatment repressed virtually all detoxification genes, with fold‑changes ranging from –2 to –10. β‑SNP produced a mixed response: GCS and GS were modestly induced, whereas GR and glutathione‑S‑transferase (GST) were strongly down‑regulated. Genes of the thioredoxin/glutaredoxin (Trx/Grx) network were either undetectable or markedly reduced after SNP exposure, indicating a broad suppression of antioxidant defenses.

Enzymatic assays of GR activity revealed a biphasic response to increasing SNP concentrations: low concentrations modestly stimulated activity, whereas higher concentrations caused pronounced inhibition. When iodoacetic acid (IAA), a known GSH‑depleting agent, was added, GR activity was completely abolished, and the MICs of both SNP types fell dramatically. This demonstrates that SNPs are particularly lethal when the glutathione pool is compromised, and that the modest GR activation observed at SICs is insufficient to detoxify the nanoparticles.

The authors propose that nanoscale dimensions combined with polymeric surface coatings enable SNPs to evade the proactive GR‑mediated detox pathway that efficiently processes bulk sulfur. Instead, the particles penetrate the mitochondrial matrix, disrupt membrane integrity, and inhibit cytochrome‑c oxidase (complex IV), thereby impairing oxidative phosphorylation. The transcriptomic repression of GSH‑GSSG and Trx/Grx genes suggests that SNPs also interfere with the transcriptional regulation of antioxidant enzymes, further weakening fungal defenses.

In conclusion, this work provides the first comprehensive transcriptomic and enzymatic evidence that sulfur nanoparticles can bypass the glutathione reductase detox system in fungi. Their nanometric size and polymeric surface modification confer a mode of action distinct from elemental sulfur, making them promising candidates for next‑generation antifungal agents with a reduced likelihood of resistance development. Future studies should extend these findings to metabolomic profiling, in‑vivo plant protection assays, and safety assessments for potential agricultural or clinical applications.