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At least 19 recordsLinked to original sources
PubMed1999

Determination of optimum growth conditions for gliotoxin production by Aspergillus fumigatus and development of a novel method for gliotoxin detection.

Gliotoxin is a toxic metabolite of Aspergillus fumigatus Fresenius and other fungi. It has been suggested that this toxin may play an important role in the pathogenesis of aspergillosis as gliotoxin has immunosuppressive activity both in vitro and in vivo. We have determined the optimum growth conditions for the production of gliotoxin by selected isolates of A. fumigatus using a number of defined media. Gliotoxin was detected by thin layer chromatography and high performance liquid chromatography. The carbohydrate source, concentration of carbohydrate in the growth medium and incubation temperature were all found to influence gliotoxin production. Optimum growth conditions for gliotoxin production in our study were Czapek-Dox broth containing 30% glucose and incubation at 37 degrees C. Most of the gliotoxin was produced after 29 h incubation, during the exponential phase of growth. A novel method for screening large numbers of A. fumigatus isolates for gliotoxin production, which is both quick and easy, has also been developed, based on the ability of gliotoxin to inhibit the adherence of lung fibroblast (L929) cells to plastic microtitre plates.

Animals↗
PubMed2000

Gliotoxin-induced cytotoxicity proceeds via apoptosis and is mediated by caspases and reactive oxygen species in LLC-PK1 cells.

Renal failure associated with aspergillosis is caused by pathogenic fungi. Gliotoxin is a toxic epipolythiodioxopiperazine metabolite produced by the pathogens. The present study investigated the cytotoxicity and underlying mechanisms induced by gliotoxin in LLC-PK1 cells, a porcine renal proximal tubular cell line. Gliotoxin at 100 ng/ml did not show a cytotoxic effect, but unmasked a dose-dependent cell death induced by TNF-alpha. TNF-alpha-induced cell death in the presence of gliotoxin was associated with hypodiploid nuclei and activation of caspase-3-like proteases. Blockade of caspases by boc-aspartyl (OMe)-fluoromethylketone and z-DEVD.fmk inhibited TNF-alpha-induced cell death. As the concentrations of gliotoxin were increased, gliotoxin killed the cells directly in a dose-dependent manner. Further analyses of DNA fragmentation, hypodiploid nuclei, mitochondrial membrane potential, and plasma membrane integrity revealed that cell death proceeded via apoptosis. Gliotoxin-induced apoptosis was associated with dose-dependent and time-dependent activation of caspase-3-like proteases. Boc-aspartyl (OMe)-fluoromethylketone attenuated the killing effect. Gliotoxin also increased the intracellular levels of reactive oxygen species as measured by flow cytometry. N-acetylcysteine, a well-known antioxidant, completely abolished the gliotoxin-induced caspase-3-like activity, cytotoxicity, and reactive oxygen species. In conclusion, (1) gliotoxin at 100 ng/ml unmasks the ability of TNF-alpha-induced apoptosis, and the effect of TNF-alpha is mediated by caspase-3-like proteases; and (2) at higher concentrations gliotoxin itself induces cell death, which is via apoptosis and dependent on caspase-3-like activity and reactive oxygen species.

Animals↗
PubMed2002

Gliotoxin, an inhibitor of nuclear factor-kappa B, attenuates peptidoglycan-polysaccharide-induced colitis in rats.

Gliotoxin is a fungal metabolite that has immunosuppressive properties. First, we determined if gliotoxin could inhibit bacterial peptidoglycan-polysaccharide-stimulated tumor necrosis factor-alpha production, as well as nuclear factor-kappa B (NF-kappaB), in a rat macrophage (NR8383) cell line. Next, the apoptosis-inducing potential of gliotoxin was also evaluated in this cell line. Finally, we evaluated whether gliotoxin could reduce peptidoglycan-polysaccharide-induced colitis in rats. Gliotoxin (2 mg/kg/day) was dosed from day 14 after the initial intramural colonic injection of peptidoglycan-polysaccharide until day 21. A gross colonic injury score, myeloperoxidase activity, and cytokine levels were all evaluated on day 21. Gliotoxin dose dependently inhibited cytokine production, as well as NF-kappaB, and also induced apoptosis in the NR8383 cell line. On day 21, gliotoxin significantly reduced gross colonic injury (adhesions, nodules, mucosal lesions) in rats. Gliotoxin-treated rats also had partially normalized biochemical indices of colitis, such as colonic cytokine levels. The colonic level of NF-kappaB was also partially normalized in gliotoxin treated rats. Gliotoxin also exhibited an antiarthritis effect in peptidoglycan-polysaccharide-treated rats. In summary, gliotoxin effectively attenuated the chronic reactivation phase of peptidoglycan-polysaccharide-induced colitis. This anticolitis effect may be related to the inhibition of NF-kappaB in Lewis rats.

Animals↗
PubMed1994

Cellular uptake and release of the immunomodulating fungal toxin gliotoxin.

Uptake of the immunomodulating agent gliotoxin into a panel of cells using biosynthetically radiolabelled 35S toxin showed rapid association of the toxin with all cell types studied with 70-85% of the total counts in the media becoming cell associated. A difference in kinetics was observed for cell lines when compared to the primary cells thymocytes, activated T-cells and macrophages. In the latter uptake was maximal after 10-15 min and radiolabel was lost from the cells as early as 100 min. In the cell lines studied, uptake was complete in less than 1 min with no loss of label after 100 min. The exception to this was a Wilms tumour line. Analysis of the fate of gliotoxin taken up into sensitive (activated T-cells) and resistant (human fibroblast) cells by HPLC showed: (a) up to 30% of the original gliotoxin taken up by sensitive cells was released as free gliotoxin over a 22 hr period. The remainder was metabolized to inorganic sulphate; (b) in T-cells gliotoxin is reduced to the dithiol form in significant amounts and this reduction may be modulated by glutathione; and (c) no reduced gliotoxin could be detected in the resistant fibroblast cell line 27Sk even though up to 50% of the original gliotoxin was still present in the free form in these cells at 22 hr. Gliotoxin became covalently associated with macromolecules in both cell types studied. Very little free gliotoxin is released into extracellular medium by the fibroblast cell line. Gliotoxin at 500 nM was found to induce apoptosis or programmed cell death in the Wilms tumour cell line but not in any other cell line studied, and this may account for the different kinetics of release of the toxin from the Wilms tumour cell line.

Animals↗
PubMed2006

Gliotoxin reduces the severity of trinitrobenzene sulfonic acid-induced colitis in mice: evidence of the connection between heme oxygenase-1 and the nuclear factor-kappaB pathway in vitro and in vivo.

BACKGROUND: Gliotoxin, a fungal metabolite, has been known to show strong immunosuppressive properties, although its mechanisms are not completely understood. In this report, the authors investigated the mechanism whereby gliotoxin has anti-inflammatory properties in vitro and in trinitrobenzene sulfonic acid-induced colitis. MATERIALS AND METHODS: Body weight, histological scores, and myeloperoxidase activity were evaluated in trinitrobenzene sulfonic acid colitis. Nuclear factor-kappaB (NF-kappaB) p65, tumor necrosis factor-alpha, interleukin (IL)-1beta, IL-12, and intercellular adhesion molecule-1 were detected by immunohistochemical staining. IL-8 secretion was measured by an enzyme-linked immunosorbent assay. Heme oxygenase-1 (HO-1) expression and I-kappaB degradation were analyzed by Western blot. RESULTS: Pretreatment of human epithelial HT-29 cells with gliotoxin significantly blocked the I-kappaB degradation and NF-kappaB p65 nuclear translocation induced by tumor necrosis factor-alpha or IL-1beta; these were parallel with the inhibition of IL-8 secretion and intercellular adhesion molecule-1 expression in the same cells. Interestingly, gliotoxin induced HO-1 in HT-29 cells and, in turn, inhibition of HO-1 activity by a zinc protoporphyrin IX reversed the effects of gliotoxin in terms of I-kappaB degradation, intercellular adhesion molecule-1 expression, and IL-8 production. In trinitrobenzene sulfonic acid colitis, gliotoxin administration significantly improved the clinical and histopathological symptoms. Notably, gliotoxin also induced HO-1 in the colonic mucosa and zinc protoporphyrin IX reversed the protective effects of gliotoxin in trinitrobenzene sulfonic acid colitis. CONCLUSIONS: These results demonstrate for the first time that the anti-inflammatory actions mediated by gliotoxin include HO-1 induction and the subsequent blockade of NF-kappaB-dependent signaling pathways in vitro and in vivo. The current results also demonstrate that gliotoxin may be an effective agent for the treatment of diseases characterized by mucosal inflammation.

Animals↗
PubMed2004

Mechanism of action of the antifibrogenic compound gliotoxin in rat liver cells.

Gliotoxin has been shown to promote a reversal of liver fibrosis in an animal model of the disease although its mechanism of action in the liver is poorly defined. The effects of gliotoxin on activated hepatic stellate cells (HSCs) and hepatocytes have therefore been examined. Addition of gliotoxin (1.5 microM) to culture-activated HSCs resulted in its rapid accumulation, resulting in increased levels of glutathione and apoptosis without any evidence of oxidative stress. In contrast, although hepatocytes also rapidly sequestered gliotoxin, cell death only occurred at high (50-microM) concentrations of gliotoxin and by necrosis. At high concentrations, gliotoxin was metabolized by hepatocytes to a reduced (dithiol) metabolite and glutathione was rapidly oxidized. Fluorescent dye loading experiments showed that gliotoxin caused oxidative stress in hepatocytes. Antioxidants--but not thiol redox active compounds--inhibited both oxidative stress and necrosis in hepatocytes. In contrast, HSC apoptosis was not affected by antioxidants but was potently abrogated by thiol redox active compounds. The adenine nucleotide transporter (ANT) is implicated in mitochondrial-dependent apoptosis. HSCs expressed predominantly nonliver ANT isoform 1, and gliotoxin treatment resulted in a thiol redox-dependent alteration in ANT mobility in HSC extracts, but not hepatocyte extracts. In conclusion, these data suggest that gliotoxin stimulates the apoptosis of HSCs through a specific thiol redox-dependent interaction with the ANT. Further understanding of this mechanism of cell death will aid in finding therapeutics that specifically stimulate HSC apoptosis in the liver, a promising approach to antifibrotic therapy.

Animals↗
PubMed2000

In vitro and in vivo effects of gliotoxin, a fungal metabolite: efficacy against dextran sodium sulfate-induced colitis in rats.

Gliotoxin is a fungal metabolite that has immunosuppressive properties. First, we determined if gliotoxin could inhibit cytokine production from macrophage and colonic epithelial cell lines, as well as whether it inhibited nuclear factor-kappa B in these same cell types. Second, we evaluated whether gliotoxin could reduce dextran sodium sulfate-induced colitis in rats. A disease activity index, myeloperoxidase activity, and cytokine levels were evaluated on either day 7 or 21. In both cell lines, gliotoxin dose dependently inhibited cytokine production and nuclear factor-kappa B. On day 21, gliotoxin significantly reduced disease activity (diarrhea and bloody stools) in rats. On day 7, gliotoxin treatment significantly improved various indices of colitis, including colonic cytokine levels. Decreased food consumption and weight gain was evident with a larger dose of gliotoxin. In summary, gliotoxin, a nuclear factor-kappa B inhibitor, effectively reduced dextran sodium sulfate-induced colitis in rats. However, gliotoxin exhibited a narrow therapeutic to toxicity ratio in these rats.

Animals↗
PubMed2006

Disruption of a nonribosomal peptide synthetase in Aspergillus fumigatus eliminates gliotoxin production.

The fungal secondary metabolite gliotoxin produced by Aspergillus fumigatus has been hypothesized to be important in the development of invasive aspergillosis. In this study, we addressed this hypothesis by disrupting a nonribosomal peptide synthetase (NRPS) (encoded by gliP) predicted to be involved in gliotoxin production. Mutants with a disrupted gliP locus failed to produce gliotoxin, which confirmed the role of the NRPS encoded by gliP in gliotoxin biosynthesis. We found no morphological, developmental, or physiological defects in DeltagliP mutant strains. In addition, disruption of gliP resulted in down regulation of gene expression in the gliotoxin biosynthesis gene cluster, which was restored with addition of exogenous gliotoxin. This interesting result suggests a role for gliotoxin in regulating its own production. Culture filtrates from the DeltagliP mutant were unable to inhibit ionomycin-dependent degranulation of mast cells, suggesting a role for gliotoxin in suppressing mast cell degranulation and possibly in disease development. However, the DeltagliP mutant did not have an impact on survival or tissue burden in a murine inhalational model of invasive aspergillosis. This result suggests that gliotoxin is not required for virulence in an immunosuppressed host with an invasive pulmonary infection.

Animals↗
PubMed2005

Influence of media and temperature on gliotoxin production in Aspergillus fumigatus strains.

Gliotoxin is a secondary metabolite of the epipolythiodioxopiperazine family with biologically active internal disulfide bridge. It is produced by many fungal species, including Aspergillus fumigatus and A. terreus. A. fumigatus, which produces gliotoxin and more than twenty other secondary metabolites, is the leading cause of invasive aspergillosis. Gliotoxin production in situ influence the development of aspergillosis. This study investigated the in vitro production of gliotoxin in nine A. fumigatus isolates from the upper respiratory tract of immunocompromised patients. The effects of media composition and incubation temperature were studied. Gliotoxin was extracted from biomass and its concentration was semi-quantitatively analysed using thin-layer chromatography. Gliotoxin production was higher in the yeast-extract liquid medium (YES) than in the synthetic Czapek-Dox liquid medium (CZA). Incubation at 37 degrees C resulted in higher gliotoxin production than at 25 degrees C, probably because higher temperatures favour expansive growth of the mycelium. Gliotoxin could be detected after three days of incubation at concentrations 4.06 mg mL(-1) (in YES at 37 degrees C) and 1.07 mg mL(-1) (in CZA at 25 degrees C). YES broth as a medium containing 4% sucrose and 2% of yeast extract is a very rich substrate for the production of gliotoxin in vitro.

Aspergillus fumigatus↗
PubMed2006

Glutathione intensifies gliotoxin-induced cytotoxicity in human neuroblastoma SH-SY5Y cells.

Gliotoxin is a fungal second metabolite produced by diverse species that can be found in compost, stored crops, moist animal feed and sawdust. The role of glutathione in gliotoxin-induced toxicity was studied in order to elucidate the toxic mechanisms leading to neurite degeneration and cell death in differentiated human neuroblastoma (SH-SY5Y) cells. After 72 h of exposure to gliotoxin, moderate cytotoxicity was induced at 0.1 micromol/L, which was more severe at higher concentrations. A reduction in the number of neurites per cell was also observed. By decreasing the level of intracellular glutathione with L: -buthionine-sulfoxamine (BSO) a specific inhibitor of glutathione synthesis, the cytotoxic effect of gliotoxin was significantly attenuated. The gliotoxin-induced cytotoxicity was also slightly reduced by the antioxidant vitamin C. However, the neurite degenerative effect was not altered by BSO, or by vitamin C. A concentration-dependent increase in the ratio between oxidized and reduced forms of glutathione, as well as the total intracellular glutathione levels, was noted after exposure to gliotoxin. The increase of glutathione was also reflected in western blot analyses showing a tendency for the regulatory subunit of gamma-glutamylcysteine synthetase to be upregulated. In addition, the activity of glutathione reductase was slightly increased in gliotoxin-exposed cells. These results indicate that glutathione promotes gliotoxin-induced cytotoxicity, probably by reducing the ETP (epipolythiodioxopiperazine) disulfide bridge to the dithiol form.

Cell Line, Tumor↗
PubMed2006

A new and rapid bioassay for the detection of gliotoxin and related epipolythiodioxopiperazines produced by fungi.

Gliotoxin is an immunosuppressive cytotoxin produced by numerous environmental or pathogenic fungal species. For this reason, it is one of the mycotoxins which must be systematically searched for in samples for biological control. In this study, a new, rapid and sensitive method for detecting gliotoxin has been developed. This bioassay is based on the induction of morphological changes in cultured cells (human KB cell line) by gliotoxin. Interpretation of the assay can be carried out after 1 h of incubation, either by direct microscopic observation, or with an automated microplate-reader at 630 nm. The limit of detection is 18-20 ng of gliotoxin in the well, depending on the used observation method. A high degree of specificity of the detection is brought about by the ability of the reducing reactant dithiothreitol to inhibit the biological activities of epipolythiodioxopiperazines (ETPs), such as gliotoxin, by reducing their polysulfide bridge. The bioassay allows a rapid primary screening of samples and a semi-quantitative evaluation of the gliotoxin concentration in extracts. The method has been used to study the gliotoxin production by different fungal strains, allowing to highlight 3 strains of Aspergillus fumigatus producing gliotoxin in various extracts.

Aspergillus fumigatus↗
PubMed2000

The fungal metabolite gliotoxin: immunosuppressive activity on CTL-mediated cytotoxicity.

Gliotoxin, a potential etiologic agent which is synthesized by Aspergillus fumigatus and other pathogenic fungi, exhibits a variety of immunosuppressive activities. We have found that gliotoxin markedly inhibits both perforin-dependent and Fas ligand-dependent cytotoxic T-lymphocyte (CTL)-mediated cytotoxicity. Gliotoxin blocked granule exocytosis and the production of inositol phosphates in response to anti-CD3 stimulation. Apparently, activation signals were not efficiently received by the gliotoxin-treated CTL clone, perhaps because gliotoxin profoundly disturbed CTL cell attachment to immobilized anti-CD3. Although the expression of surface molecules of the CTL clone such as CD3 was unaffected by gliotoxin, the effector/target conjugate formation was inhibited dose-dependently by gliotoxin treatment of the effector CTL clone. These results suggest that gliotoxin prevents CTL from interacting with target cells.

Animals↗
PubMed1999

The secondary fungal metabolite gliotoxin targets proteolytic activities of the proteasome.

BACKGROUND: The fungal epipolythiodioxopiperazine metabolite gliotoxin has a variety of toxic effects such as suppression of antigen processing, induction of macrophagocytic apoptosis and inhibition of transcription factor NF-kappaB activation. How gliotoxin acts remains poorly understood except that the molecule's characteristic disulfide bridge is important for immunomodulation. As this fungal metabolite stabilizes the NF-kappaB inhibitor IkappaBalpha in the cytoplasm, we decided to investigate its molecular mechanism of action. RESULTS: We show that gliotoxin is an efficient, noncompetitive inhibitor of the chymotrypsin-like activity of the 20S proteasome in vitro. Proteasome inhibition can be reversed by dithiothreitol, which reduces gliotoxin to the dithiol compound. In intact cells, gliotoxin inhibits NF-kappaB induction through inhibition of proteasome-mediated degradation of IkappaBalpha. CONCLUSIONS: Gliotoxin targets catalytic activities of the proteasome efficiently. Inhibition by gliotoxin may be countered by reducing agents, which are able to inactivate the disulfide bridge responsible for the inhibitory capacity of gliotoxin.

Cells, Cultured↗
PubMed1997

Apoptosis induced by gliotoxin is preceded by phosphorylation of histone H3 and enhanced sensitivity of chromatin to nuclease digestion.

The fungal toxin gliotoxin induces apoptotic cell death in a variety of cells. Apoptosis induced in thymocytes by gliotoxin is rapid, and DNA fragmentation is observable within 4 h treatment. Apoptosis induced by gliotoxin is calcium-independent and unaffected by protein synthesis inhibitors. We have previously shown that gliotoxin results in phosphorylation of a 16.3-kDa protein within 10 min treatment of thymocytes. Here we show that this protein is histone H3 and phosphorylation occurs on Ser-10. Cyclic AMP levels and activity of protein kinase A (PKA) are raised in cells treated with gliotoxin. Apoptosis is inhibited by genistein which also inhibits PKA and histone H3 phosphorylation. Apoptosis is also inhibited by a number of specific inhibitors of PKA suggesting apoptosis induced by gliotoxin is modulated by this kinase. The agents forskolin and cholera toxin do not induce rapid phosphorylation of H3 although some increase in phosphorylation of H3 does occur after 8 h with these agents. Forskolin and cholera toxin also induce apoptosis but over a longer time course than gliotoxin. In all cases levels of apoptosis correlate with degree of H3 phosphorylation. Cells treated with gliotoxin show an early sensitivity to micrococcal nuclease and DNase I digestion indicating a functional relationship between DNA fragmentation and H3 phosphorylation.

Amino Acid Sequence↗
PubMed2004

Effect and stability of gliotoxin, an Aspergillus fumigatus toxin, on in vitro rumen fermentation.

Aspergillus fumigatus is a toxicogenic fungus usually found in contaminated animal feeds, especially in conserved forages where it can produce several mycotoxins. Gliotoxin, one of the most important toxic metabolites produced by this fungus, has antibacterial, immunosuppressive and apoptotic effects. Ruminants due to the high proportion of forages they receive in the ration would be particularly exposed to gliotoxin. The objective of this work was (1) to assess the effect of gliotoxin on in vitro rumen fermentation and (2) to determine the effect of fermentation on gliotoxin stability. Gliotoxin did not affect rumen fermentation at concentrations found in naturally contaminated feeds. No effects were observed up to a concentration of 20 microg toxin ml(-1) and an extremely high toxin concentration (80 microg ml(-1)) was necessary to affect dry matter degradation, gas and total volatile fatty acids production by 24, 37 and 18%, respectively (p < 0.01). In addition, the toxin was unstable in the rumen environment with 90% disappearance at 6 h of incubation (p < 0.05). In contrast, extracts of A. fumigatus cultures containing gliotoxin at concentrations several times lower than that used for experiments with pure toxin had a negative effect on fermentations indicating the toxicity and possible synergism of other metabolites produced by this fungus. Extracts containing 8.8 microg gliotoxin ml(-1) decreased dry matter degradation, gas and volatile fatty acids production by 28, 46 and 35%, respectively (p < 0.01). Identification of these toxic metabolites and assessment of the rate of passage of gliotoxin to the lower intestinal tract is necessary to evaluate the potential risk of these toxins to ruminants.

Animal Feed↗
PubMed2006

Gliotoxin-mediated suppression of innate and adaptive immune functions directed against Listeria monocytogenes.

Gliotoxin is an immunosuppressive apoptogenic mycotoxin produced by a number of fungi including important human pathogens as Aspergillus fumigatus. In order to elucidate the potential role of gliotoxin as immunoevasive fungal virulence factor we studied the effects of gliotoxin on the innate and adaptive T cell-mediated immune response against the facultatively intracellular bacterium Listeria monocytogenes. Gliotoxin induced apoptosis of bone marrow-derived macrophages, dendritic cells and CD8 T cells in a dose- and cell type-dependent manner. In vitro the apoptogenic effect of gliotoxin correlated with a strong reduction of TNF-alpha and interleukin (IL)-12 production by dendritic cells and bone marrow-derived macrophages infected with L. monocytogenes and in the case of infected macrophages also in reduced NO-production and recognition by L. monocytogenes-specific CD8 T cells. Further gliotoxin pre-treatment of CD8 T cells reduced target cell lysis. In vivo, treatment of mice with gliotoxin increased the bacterial burden during the innate and the adaptive phase of primary L. monocytogenes infection. Taken together, these results demonstrate the suppressive effects of gliotoxin on the innate and also on the adaptive T cell-mediated antilisterial immunity.

Animals↗