Gliotoxin

Aspergillus fumigatus causes a range of human and animal diseases collectively known as aspergillosis. A. fumigatus possesses and expresses a range of genetic determinants of virulence, which facilitate colonisation and disease progression, including the secretion of mycotoxins. Gliotoxin (GT) is the best studied A. fumigatus mycotoxin with a wide range of known toxic effects that impair human immune cell function. GT is also highly toxic to A. fumigatus and this fungus has evolved self-protection mechanisms that include (i) the GT efflux pump GliA, (ii) the GT neutralising enzyme GliT, and (iii) the negative regulation of GT biosynthesis by the bis-thiomethyltransferase GtmA. The transcription factor (TF) RglT is the main regulator of GliT and this GT protection mechanism also occurs in the non-GT producing fungus A. nidulans. However, the A. nidulans genome does not encode GtmA and GliA. This work aimed at analysing the transcriptional response to exogenous GT in A. fumigatus and A. nidulans, two distantly related Aspergillus species, and to identify additional components required for GT protection. RNA-sequencing shows a highly different transcriptional response to exogenous GT with the RglT-dependent regulon also significantly differing between A. fumigatus and A. nidulans. However, we were able to observe homologs whose expression pattern was similar in both species (43 RglT-independent and 11 RglT-dependent). Based on this approach, we identified a novel RglT-dependent methyltranferase, MtrA, involved in GT protection. Taking into consideration the occurrence of RglT-independent modulated genes, we screened an A. fumigatus deletion library of 484 transcription factors (TFs) for sensitivity to GT and identified 15 TFs important for GT self-protection. Of these, the TF KojR, which is essential for kojic acid biosynthesis in Aspergillus oryzae, was also essential for virulence and GT biosynthesis in A. fumigatus, and for GT protection in A. fumigatus, A. nidulans, and A. oryzae. KojR regulates rglT, gliT, gliJ expression and sulfur metabolism in Aspergillus species. Together, this study identified conserved components required for GT protection in Aspergillus species.

Background: Gliotoxin, a secondary metabolite isolated from marine-derived Aspergillus fumigatus, has demonstrated anti-tumor properties in several cancers. Ferroptosis, a recently discovered type of programmed cell death that depends on the accumulation of iron and lipid peroxides, participates in the occurrence and development of various diseases, including cancer. A recent patent, US20200383943, has suggested that promotion of ferroptosis is a method of cancer treatment. Therefore, the development of drugs that induce ferroptosis in cancer cells would constitute a novel therapeutic approach. Objective: Gliotoxin is a natural compound which has exhibited anti-tumor properties in multiple cancers, however studies of the effect of gliotoxin on esophageal cancer are lacking. Although cancer treatment has shown great progress, including traditional surgery, chemotherapy, radiotherapy, and immunotherapy, the prognosis of esophageal cancer is still poor. Therefore, the development of new treatment approaches for esophageal cancer is necessary. Methods: The effects of gliotoxin on esophageal cancer cells were determined by functional assays, such as CCK-8, wound healing and transwell assays. We used online tools to predict the target genes of gliotoxin, followed by further verification using Western blotting assays. To assess the role of gliotxin in inducing ferroptosis in esophageal cancer, we detected characteristics associated with ferroptosis including ROS, MDA, GSH and Fe2+. Results: Using online tools SEA and SwissTargetPrediction, we predicted that SUV39H1 was the gliotoxin target gene. Furthermore, in esophageal cancer tissues SUV39H1 was expressed at higher levels than normal tissues, while in patients with Esophageal Squamous Cell Carcinoma (ESCC) high expression levels of SUV39H1 indicated a poor prognosis. In vitro, we observed that gliotoxin increased ESCC cell death and inhibited cell migration. We treated ESCC cells with pan-caspase inhibitor Z-VAD-FMK or ferroptosis inhibitors including Fer-1 and DFO. Our results showed that Fer-1 and DFO reduced the toxic effects of gliotoxin, while Z-VAD-FMK did not. Furthermore, gliotoxin treatment reduced tumor weight and volume in the xenograft tumor mouse model. Conclusion: In summary, our findings indicate that gliotoxin downregulated SUV39H1 expression in ESCC cells and induced ferroptosis, suggesting a novel natural therapy for ESSC.

Beneficial interaction of members of the fungal genus Trichoderma with plant roots primes the plant immune system, promoting systemic resistance to pathogen infection. Some strains of Trichoderma virens produce gliotoxin, a fungal epidithiodioxopiperazine (ETP)-type secondary metabolite that is toxic to animal cells. It induces apoptosis, prevents NF-κB activation via the inhibition of the proteasome, and has immunosuppressive properties. Gliotoxin is known to be involved in the antagonism of rhizosphere microorganisms. To investigate whether this metabolite has a role in the interaction of Trichoderma with plant roots, we compared gliotoxin-producing and nonproducing T. virens strains. Both colonize the root surface and outer layers, but they have differential effects on root growth and architecture. The responses of tomato plants to a pathogen challenge were followed at several levels: lesion development, levels of ethylene, and reactive oxygen species. The transcriptomic signature of the shoot tissue in response to root interaction with producing and nonproducing T. virens strains was monitored. Gliotoxin producers provided stronger protection against foliar pathogens, compared to nonproducing strains. This was reflected in the transcriptomic signature, which showed the induction of defense-related genes. Two markers of plant defense response, PR1 and Pti-5, were differentially induced in response to pure gliotoxin. Gliotoxin thus acts as a microbial signal, which the plant immune system recognizes, directly or indirectly, to promote a defense response. IMPORTANCE A single fungal metabolite induces far-reaching transcriptomic reprogramming in the plant, priming immune responses and defense, in contrast to its immunosuppressive effect on animal cells. While the negative effects of gliotoxin-producing Trichoderma strains on growth may be observed only under a particular set of laboratory conditions, gliotoxin-linked molecular patterns, including the potential for limited cell death, could strongly prime plant defense, even in mature soil-grown plants in which the same Trichoderma strain promotes growth.