11 research outputs found
Gliotoxin effects on fungal growth: Mechanisms and exploitation
Although initially investigated for its antifungal properties, little is actually known about the effect of
gliotoxin on Aspergillus fumigatus and other fungi. We have observed that exposure of A. fumigatus to
exogenous gliotoxin (14 lg/ml), under gliotoxin-limited growth conditions, results in significant alteration
of the expression of 27 proteins (up- and down-regulated >1.9-fold; p < 0.05) including de novo
expression of Cu, Zn superoxide dismutase, up-regulated allergen Asp f3 expression and down-regulated
catalase and a peroxiredoxin levels. Significantly elevated glutathione GSH levels (p < 0.05), along with
concomitant resistance to diamide, were evident in A. fumigatus ∆gliT, lacking gliotoxin oxidoreductase,
a gliotoxin self-protection gene. Saccharomyces cerevisiae deletents (∆sod1 and ∆yap1) were hypersensitive
to exogenous gliotoxin, while ∆gsh1 was resistant. Significant gliotoxin-mediated (5 µg/ml) growth
inhibition (p < 0.001) of Aspergillus nidulans, Aspergillus terreus, Aspergillus niger, Cochliobolus heterostrophus
and Neurospora crassa was also observed. Growth of Aspergillus flavus, Fusarium graminearum and
Aspergillus oryzae was significantly inhibited (p < 0.001) at gliotoxin (10 lg/ml), indicating differential
gliotoxin sensitivity amongst fungi. Re-introduction of gliT into A. fumigatus DgliT, at a different locus
(ctsD; AFUA_4G07040, an aspartic protease), with selection on gliotoxin, facilitated deletion of ctsD without
use of additional antibiotic selection markers. Absence of ctsD expression was accompanied by restoration
of gliT expression, and resistance to gliotoxin. Thus, we propose gliT/gliotoxin as a useful
selection marker system for fungal transformation. Finally, we suggest incorporation of gliotoxin sensitivity
assays into all future fungal functional genomic studies
Regulation of Nonribosomal Peptide Synthesis: bis-Thiomethylation Attenuates Gliotoxin Biosynthesis in Aspergillus fumigatus
Gliotoxin is a redox-active nonribosomal peptide
produced by Aspergillus fumigatus. Like many other
disulfide-containing epipolythiodioxopiperazines, a
bis-thiomethylated form is also produced. In the
case of gliotoxin, bisdethiobis(methylthio)gliotoxin
(BmGT) is formed for unknown reasons by a cryptic
enzyme. Here, we identify the S-adenosylmethionine-
dependent gliotoxin bis-thiomethyltransferase
(GtmA), which converts dithiogliotoxin to BmGT.
This activity, which is induced by exogenous gliotoxin,
is only detectable in protein lysates of A. fumigatus
deficient in the gliotoxin oxidoreductase, gliT.
Thus, GtmA is capable of substrate bis-thiomethylation.
Deletion of gtmA completely abrogates BmGT
formation and we now propose that the purpose
of BmGT formation is primarily to attenuate gliotoxin
biosynthesis. Phylogenetic analysis reveals 124
GtmA homologs within the Ascomycota phylum.
GtmA is encoded outside the gliotoxin biosynthetic
cluster and primarily serves to negatively regulate
gliotoxin biosynthesis. This mechanism of postbiosynthetic
regulation of nonribosomal peptide synthesis
appears to be quite unusual
The Aspergillus fumigatus Protein GliK Protects against Oxidative Stress and Is Essential for Gliotoxin Biosynthesis
The function of a number of genes in the gliotoxin biosynthetic cluster (gli) in Aspergillus fumigatus remains unknown. Here, we
demonstrate that gliK deletion from two strains of A. fumigatus completely abolished gliotoxin biosynthesis. Furthermore,
exogenous H2O2 (1 mM), but not gliotoxin, significantly induced A. fumigatus gliK expression (P 0.0101). While both mutants
exhibited significant sensitivity to both exogenous gliotoxin (P<0.001) and H2O2 (P<0.01), unexpectedly, exogenous
gliotoxin relieved H2O2-induced growth inhibition in a dose-dependent manner (0 to 10 g/ml). Gliotoxin-containing organic
extracts derived from A. fumigatus ATCC 26933 significantly inhibited (P<0.05) the growth of the gliK26933 deletion mutant.
The A. fumigatus gliK26933 mutant secreted metabolites, devoid of disulfide linkages or free thiols, that were detectable by reverse-
phase high-performance liquid chromatography and liquid chromatography-mass spectrometry with m/z 394 to 396.
These metabolites (m/z 394 to 396) were present at significantly higher levels in the culture supernatants of the A. fumigatus
gliK26933 mutant than in those of the wild type (P0.0024 [fold difference, 24] and P0.0003 [fold difference, 9.6], respectively)
and were absent from A. fumigatus gliG. Significantly elevated levels of ergothioneine were present in aqueous mycelial
extracts of the A. fumigatus gliK26933 mutant compared to the wild type (P<0.001). Determination of the gliotoxin uptake rate
revealed a significant difference (P0.0045) between that of A. fumigatus ATCC 46645 (9.3 pg/mg mycelium/min) and the
gliK46645 mutant (31.4 pg/mg mycelium/min), strongly suggesting that gliK absence and the presence of elevated ergothioneine
levels impede exogenously added gliotoxin efflux. Our results confirm a role for gliK in gliotoxin biosynthesis and reveal new
insights into gliotoxin functionality in A. fumigatus
Resistance is not futile: gliotoxin biosynthesis, functionality and utility
Gliotoxin biosynthesis is encoded by the gli gene cluster in Aspergillus fumigatus. The biosynthesis of gliotoxin is influenced by a suite of transcriptionally-active regulatory proteins and a bis-thiomethyltransferase. A self-protection system against gliotoxin is present in A. fumigatus. Several additional metabolites are also produced via the gliotoxin biosynthetic pathway. Moreover, the biosynthesis of unrelated natural products appears to be influenced either by gliotoxin or by the activity of specific reactions within the biosynthetic pathway. The activity of gliotoxin against animal cells and fungi, often mediated by interference with redox homeostasis or protein modification, is revealing new metabolic interactions within eukaryotic systems. Nature has provided a most useful natural product with which to reveal some of its many molecular secret
Gliotoxin effects on fungal growth: Mechanisms and exploitation
Although initially investigated for its antifungal properties, little is actually known about the effect of
gliotoxin on Aspergillus fumigatus and other fungi. We have observed that exposure of A. fumigatus to
exogenous gliotoxin (14 lg/ml), under gliotoxin-limited growth conditions, results in significant alteration
of the expression of 27 proteins (up- and down-regulated >1.9-fold; p < 0.05) including de novo
expression of Cu, Zn superoxide dismutase, up-regulated allergen Asp f3 expression and down-regulated
catalase and a peroxiredoxin levels. Significantly elevated glutathione GSH levels (p < 0.05), along with
concomitant resistance to diamide, were evident in A. fumigatus ∆gliT, lacking gliotoxin oxidoreductase,
a gliotoxin self-protection gene. Saccharomyces cerevisiae deletents (∆sod1 and ∆yap1) were hypersensitive
to exogenous gliotoxin, while ∆gsh1 was resistant. Significant gliotoxin-mediated (5 µg/ml) growth
inhibition (p < 0.001) of Aspergillus nidulans, Aspergillus terreus, Aspergillus niger, Cochliobolus heterostrophus
and Neurospora crassa was also observed. Growth of Aspergillus flavus, Fusarium graminearum and
Aspergillus oryzae was significantly inhibited (p < 0.001) at gliotoxin (10 lg/ml), indicating differential
gliotoxin sensitivity amongst fungi. Re-introduction of gliT into A. fumigatus DgliT, at a different locus
(ctsD; AFUA_4G07040, an aspartic protease), with selection on gliotoxin, facilitated deletion of ctsD without
use of additional antibiotic selection markers. Absence of ctsD expression was accompanied by restoration
of gliT expression, and resistance to gliotoxin. Thus, we propose gliT/gliotoxin as a useful
selection marker system for fungal transformation. Finally, we suggest incorporation of gliotoxin sensitivity
assays into all future fungal functional genomic studies
Gliotoxin effects on fungal growth: Mechanisms and exploitation
Although initially investigated for its antifungal properties, little is actually known about the effect of
gliotoxin on Aspergillus fumigatus and other fungi. We have observed that exposure of A. fumigatus to
exogenous gliotoxin (14 lg/ml), under gliotoxin-limited growth conditions, results in significant alteration
of the expression of 27 proteins (up- and down-regulated >1.9-fold; p < 0.05) including de novo
expression of Cu, Zn superoxide dismutase, up-regulated allergen Asp f3 expression and down-regulated
catalase and a peroxiredoxin levels. Significantly elevated glutathione GSH levels (p < 0.05), along with
concomitant resistance to diamide, were evident in A. fumigatus ∆gliT, lacking gliotoxin oxidoreductase,
a gliotoxin self-protection gene. Saccharomyces cerevisiae deletents (∆sod1 and ∆yap1) were hypersensitive
to exogenous gliotoxin, while ∆gsh1 was resistant. Significant gliotoxin-mediated (5 µg/ml) growth
inhibition (p < 0.001) of Aspergillus nidulans, Aspergillus terreus, Aspergillus niger, Cochliobolus heterostrophus
and Neurospora crassa was also observed. Growth of Aspergillus flavus, Fusarium graminearum and
Aspergillus oryzae was significantly inhibited (p < 0.001) at gliotoxin (10 lg/ml), indicating differential
gliotoxin sensitivity amongst fungi. Re-introduction of gliT into A. fumigatus DgliT, at a different locus
(ctsD; AFUA_4G07040, an aspartic protease), with selection on gliotoxin, facilitated deletion of ctsD without
use of additional antibiotic selection markers. Absence of ctsD expression was accompanied by restoration
of gliT expression, and resistance to gliotoxin. Thus, we propose gliT/gliotoxin as a useful
selection marker system for fungal transformation. Finally, we suggest incorporation of gliotoxin sensitivity
assays into all future fungal functional genomic studies
Genomic and Proteomic Dissection of the Ubiquitous Plant Pathogen, Armillaria mellea: Toward a New Infection Model System
Armillaria mellea is a major plant pathogen. Yet, no large-scale “-omics” data are
available to enable new studies, and limited experimental models are available to investigate
basidiomycete pathogenicity. Here we reveal that the A. mellea genome comprises 58.35 Mb,
contains 14473 gene models, of average length 1575 bp (4.72 introns/gene). Tandem mass
spectrometry identified 921 mycelial (n = 629 unique) and secreted (n = 183 unique) proteins.
Almost 100 mycelial proteins were either species-specific or previously unidentified at the protein
level. A number of proteins (n = 111) was detected in both mycelia and culture supernatant extracts.
Signal sequence occurrence was 4-fold greater for secreted (50.2%) compared to mycelial (12%)
proteins. Analyses revealed a rich reservoir of carbohydrate degrading enzymes, laccases, and lignin
peroxidases in the A. mellea proteome, reminiscent of both basidiomycete and ascomycete
glycodegradative arsenals. We discovered that A. mellea exhibits a specific killing effect against
Candida albicans during coculture. Proteomic investigation of this interaction revealed the unique
expression of defensive and potentially offensive A. mellea proteins (n = 30). Overall, our data reveal
new insights into the origin of basidiomycete virulence and we present a new model system for further studies aimed at
deciphering fungal pathogenic mechanisms
Gliotoxin effects on fungal growth: Mechanisms and exploitation
Although initially investigated for its antifungal properties, little is actually known about the effect of
gliotoxin on Aspergillus fumigatus and other fungi. We have observed that exposure of A. fumigatus to
exogenous gliotoxin (14 lg/ml), under gliotoxin-limited growth conditions, results in significant alteration
of the expression of 27 proteins (up- and down-regulated >1.9-fold; p < 0.05) including de novo
expression of Cu, Zn superoxide dismutase, up-regulated allergen Asp f3 expression and down-regulated
catalase and a peroxiredoxin levels. Significantly elevated glutathione GSH levels (p < 0.05), along with
concomitant resistance to diamide, were evident in A. fumigatus ∆gliT, lacking gliotoxin oxidoreductase,
a gliotoxin self-protection gene. Saccharomyces cerevisiae deletents (∆sod1 and ∆yap1) were hypersensitive
to exogenous gliotoxin, while ∆gsh1 was resistant. Significant gliotoxin-mediated (5 µg/ml) growth
inhibition (p < 0.001) of Aspergillus nidulans, Aspergillus terreus, Aspergillus niger, Cochliobolus heterostrophus
and Neurospora crassa was also observed. Growth of Aspergillus flavus, Fusarium graminearum and
Aspergillus oryzae was significantly inhibited (p < 0.001) at gliotoxin (10 lg/ml), indicating differential
gliotoxin sensitivity amongst fungi. Re-introduction of gliT into A. fumigatus DgliT, at a different locus
(ctsD; AFUA_4G07040, an aspartic protease), with selection on gliotoxin, facilitated deletion of ctsD without
use of additional antibiotic selection markers. Absence of ctsD expression was accompanied by restoration
of gliT expression, and resistance to gliotoxin. Thus, we propose gliT/gliotoxin as a useful
selection marker system for fungal transformation. Finally, we suggest incorporation of gliotoxin sensitivity
assays into all future fungal functional genomic studies
The Aspergillus fumigatus Protein GliK Protects against Oxidative Stress and Is Essential for Gliotoxin Biosynthesis
The function of a number of genes in the gliotoxin biosynthetic cluster (gli) in Aspergillus fumigatus remains unknown. Here, we
demonstrate that gliK deletion from two strains of A. fumigatus completely abolished gliotoxin biosynthesis. Furthermore,
exogenous H2O2 (1 mM), but not gliotoxin, significantly induced A. fumigatus gliK expression (P 0.0101). While both mutants
exhibited significant sensitivity to both exogenous gliotoxin (P<0.001) and H2O2 (P<0.01), unexpectedly, exogenous
gliotoxin relieved H2O2-induced growth inhibition in a dose-dependent manner (0 to 10 g/ml). Gliotoxin-containing organic
extracts derived from A. fumigatus ATCC 26933 significantly inhibited (P<0.05) the growth of the gliK26933 deletion mutant.
The A. fumigatus gliK26933 mutant secreted metabolites, devoid of disulfide linkages or free thiols, that were detectable by reverse-
phase high-performance liquid chromatography and liquid chromatography-mass spectrometry with m/z 394 to 396.
These metabolites (m/z 394 to 396) were present at significantly higher levels in the culture supernatants of the A. fumigatus
gliK26933 mutant than in those of the wild type (P0.0024 [fold difference, 24] and P0.0003 [fold difference, 9.6], respectively)
and were absent from A. fumigatus gliG. Significantly elevated levels of ergothioneine were present in aqueous mycelial
extracts of the A. fumigatus gliK26933 mutant compared to the wild type (P<0.001). Determination of the gliotoxin uptake rate
revealed a significant difference (P0.0045) between that of A. fumigatus ATCC 46645 (9.3 pg/mg mycelium/min) and the
gliK46645 mutant (31.4 pg/mg mycelium/min), strongly suggesting that gliK absence and the presence of elevated ergothioneine
levels impede exogenously added gliotoxin efflux. Our results confirm a role for gliK in gliotoxin biosynthesis and reveal new
insights into gliotoxin functionality in A. fumigatus