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Regulation of sulphur assimilation is essential for virulence and affects iron homeostasis of the human-pathogenic mould Aspergillus fumigatus.

Amich J, Schafferer L, Haas H, Krappmann S - PLoS Pathog. (2013)

Bottom Line: Surprisingly, A. fumigatus is able to utilize volatile sulphur compounds produced by its methionine catabolism, a process that has not been described before and that is MetR-dependent.The A. fumigatus MetR transcriptional activator is important for virulence in both leukopenic mice and an alternative mini-host model of aspergillosis, as it was essential for the development of pulmonary aspergillosis and supported the systemic dissemination of the fungus.Taken together, this study provides evidence that regulation of sulphur assimilation is not only crucial for A. fumigatus virulence but also affects the balance of iron in this prime opportunistic pathogen.

View Article: PubMed Central - PubMed

Affiliation: Research Center for Infectious Diseases, Julius-Maximilians-University Würzburg, Würzburg, Germany.

ABSTRACT
Sulphur is an essential element that all pathogens have to absorb from their surroundings in order to grow inside their infected host. Despite its importance, the relevance of sulphur assimilation in fungal virulence is largely unexplored. Here we report a role of the bZIP transcription factor MetR in sulphur assimilation and virulence of the human pathogen Aspergillus fumigatus. The MetR regulator is essential for growth on a variety of sulphur sources; remarkably, it is fundamental for assimilation of inorganic S-sources but dispensable for utilization of methionine. Accordingly, it strongly supports expression of genes directly related to inorganic sulphur assimilation but not of genes connected to methionine metabolism. On a broader scale, MetR orchestrates the comprehensive transcriptional adaptation to sulphur-starving conditions as demonstrated by digital gene expression analysis. Surprisingly, A. fumigatus is able to utilize volatile sulphur compounds produced by its methionine catabolism, a process that has not been described before and that is MetR-dependent. The A. fumigatus MetR transcriptional activator is important for virulence in both leukopenic mice and an alternative mini-host model of aspergillosis, as it was essential for the development of pulmonary aspergillosis and supported the systemic dissemination of the fungus. MetR action under sulphur-starving conditions is further required for proper iron regulation, which links regulation of sulphur metabolism to iron homeostasis and demonstrates an unprecedented regulatory crosstalk. Taken together, this study provides evidence that regulation of sulphur assimilation is not only crucial for A. fumigatus virulence but also affects the balance of iron in this prime opportunistic pathogen.

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Related in: MedlinePlus

Regulatory cross-talk between sulphur and iron metabolism.(A) Fungal cultures were shifted after prolonged growth in medium containing methionine as sole source of sulphur [Met (22 h)] to medium depleted for this amino acid but containing sulphate, and samples for RNA preparation were taken after the indicated time points. Northern hybridisation expression analysis of several genes directly related with iron metabolism was carried out. Although there is sufficient iron in the medium (100 µM), after eight hours of incubation in media containing sulphate (constituting sulphur starving conditions for the mutant) increased transcription of genes typically expressed under iron-depleted conditions (mirB: siderophore transporter; hapX: transcriptional activator of the iron regulon; sidA: L-ornithine monooxygenase; amcA: mitochondrial ornithine exporter) is observed in the metRΔ strain as well as decreased expression of genes whose products participate in iron consuming processes (cycA: cytochrome C; acoA: aconitase; isa1: mitochondrial cluster biosynthetic protein). (B) Relative quantity of ferricrocin (FC) in the respective fungal mycelia. FC levels are already elevated before the shift, and they increase nearly fivefold under sulphur starvation conditions. (C) The wild-type strain is able to grow in the presence of 10 mM of iron, a substantially high concentration that already provokes toxic effects. When sufficient methionine (5 mM) is present, the metRΔ mutant behaves as the wild-type and the reconstituted strain. However, under sulphur starvation, the mutant is not able grow on 0.5 mM of iron or higher concentrations. This hypersensitivity might be a result of the lower expression of cccA gene (encoding a vacuolar iron transporter which participates in iron detoxification) in the metRΔ mutant, especially under sulphur starvation conditions.
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ppat-1003573-g008: Regulatory cross-talk between sulphur and iron metabolism.(A) Fungal cultures were shifted after prolonged growth in medium containing methionine as sole source of sulphur [Met (22 h)] to medium depleted for this amino acid but containing sulphate, and samples for RNA preparation were taken after the indicated time points. Northern hybridisation expression analysis of several genes directly related with iron metabolism was carried out. Although there is sufficient iron in the medium (100 µM), after eight hours of incubation in media containing sulphate (constituting sulphur starving conditions for the mutant) increased transcription of genes typically expressed under iron-depleted conditions (mirB: siderophore transporter; hapX: transcriptional activator of the iron regulon; sidA: L-ornithine monooxygenase; amcA: mitochondrial ornithine exporter) is observed in the metRΔ strain as well as decreased expression of genes whose products participate in iron consuming processes (cycA: cytochrome C; acoA: aconitase; isa1: mitochondrial cluster biosynthetic protein). (B) Relative quantity of ferricrocin (FC) in the respective fungal mycelia. FC levels are already elevated before the shift, and they increase nearly fivefold under sulphur starvation conditions. (C) The wild-type strain is able to grow in the presence of 10 mM of iron, a substantially high concentration that already provokes toxic effects. When sufficient methionine (5 mM) is present, the metRΔ mutant behaves as the wild-type and the reconstituted strain. However, under sulphur starvation, the mutant is not able grow on 0.5 mM of iron or higher concentrations. This hypersensitivity might be a result of the lower expression of cccA gene (encoding a vacuolar iron transporter which participates in iron detoxification) in the metRΔ mutant, especially under sulphur starvation conditions.

Mentions: It is well established that defects in mitochondrial Fe-S cluster biogenesis or transport induce transcription of the iron regulon [58], [59] and that Fe-S cluster-containing proteins participate directly in sensing iron availability in S. cerevisiae[60], [61]. Accordingly, we expected a regulatory cross-talk between sulphur assimilation and iron homeostasis in A. fumigatus, which was further indicated by the transcriptional profiling data (see above), however, which had not been tested in eukaryotes so far. We took advantage of our metRΔ mutant strain, which can be rapidly depleted for sulphur, to test this hypothesis. Overnight grown strains were shifted from culture media containing methionine to media depleted for this amino acid but containing sulphate. These media pose sulphur starving-conditions for the deletion strain but not for the wild-type or the reconstituted strain. After eight hours of incubation, although there was sufficient iron in the medium, the mutant strain increased transcription of several genes encoding proteins that participate in iron acquisition that are known to be upregulated under iron starvation, i.e. genes involved in siderophore biosynthesis (sidA), siderophore transport (mirB), mitochondrial ornithine export (amcA) and iron regulation (hapX) [57]. In contrast, the mutant decreased transcription of genes whose products participate in iron-consuming processes that are known to be downregulated under iron depleted conditions [40], i.e. genes encoding aconitase (acoA), cytochrome c (cycA) or components of the mitochondrial iron-sulfur-cluster biosynthetic machinery (isa1) (Fig. 8A). To analyse whether the cells were indeed depleted for iron, levels of iron chelated by ferricrocin (FC), the intracellular siderophore used for iron storage and transport [62], [63], were measured (Fig. 8B). Despite an expression pattern resembling that of iron starvation, the AfS167 mutant showed a nearly fivefold increased FC content. Combining ferricrocin analysis with total intracellular iron level measurements further underscored this imbalance in iron homeostasis (Table 4): MetR deficiency raises the cellular iron content 1.6-fold in the presence of methionine, which increases to 2.8-fold in its absence. The metRΔ mutant furthermore displays a 5-fold increased FC-chelated iron content under +Met conditions that is further enhanced to 7.5-fold when this S-sources is withdrawn. In conclusion, the cells indeed contain sufficient amounts of iron but display a defect in iron sensing and/or regulation. This dysregulation, causing an enhanced expression of iron uptake-related genes under sulphur starving conditions, translates into a phenotype of hypersensitivity to iron (Fig. 8C). At a low concentration of iron and methionine the metRΔ mutant was able to grow, although poorly due to the shortage of sulphur. This phenotype was recovered with higher availability of methionine. However, at higher iron concentrations, the wild-type strain could grow while the AfS167 mutant did not, unless a high amount of methionine was present in the medium. This might be the consequence of both the inability to shut down expression of iron uptake-related genes and of the lower cccA gene expression, encoding a recently described vacuolar transporter that has a prominent role in iron detoxification [64], in the metRΔ mutant especially under sulphur starvation (Fig. 8A), which results in iron accumulation (see Table 4: 2.4-fold increase for the metRΔ deletant under –Met conditions) to presumably toxic levels in the cytosol. Dysregulation of iron homeostasis was also tested in the wild-type strain by shifting its mycelium to a medium completely depleted for sulphur. However, expression of the iron regulon was not observed even after 24 hours of incubation (not shown), most likely because of the fact that the wild-type does not face such severe sulphur starving conditions as its metRΔ derivative apparently does.


Regulation of sulphur assimilation is essential for virulence and affects iron homeostasis of the human-pathogenic mould Aspergillus fumigatus.

Amich J, Schafferer L, Haas H, Krappmann S - PLoS Pathog. (2013)

Regulatory cross-talk between sulphur and iron metabolism.(A) Fungal cultures were shifted after prolonged growth in medium containing methionine as sole source of sulphur [Met (22 h)] to medium depleted for this amino acid but containing sulphate, and samples for RNA preparation were taken after the indicated time points. Northern hybridisation expression analysis of several genes directly related with iron metabolism was carried out. Although there is sufficient iron in the medium (100 µM), after eight hours of incubation in media containing sulphate (constituting sulphur starving conditions for the mutant) increased transcription of genes typically expressed under iron-depleted conditions (mirB: siderophore transporter; hapX: transcriptional activator of the iron regulon; sidA: L-ornithine monooxygenase; amcA: mitochondrial ornithine exporter) is observed in the metRΔ strain as well as decreased expression of genes whose products participate in iron consuming processes (cycA: cytochrome C; acoA: aconitase; isa1: mitochondrial cluster biosynthetic protein). (B) Relative quantity of ferricrocin (FC) in the respective fungal mycelia. FC levels are already elevated before the shift, and they increase nearly fivefold under sulphur starvation conditions. (C) The wild-type strain is able to grow in the presence of 10 mM of iron, a substantially high concentration that already provokes toxic effects. When sufficient methionine (5 mM) is present, the metRΔ mutant behaves as the wild-type and the reconstituted strain. However, under sulphur starvation, the mutant is not able grow on 0.5 mM of iron or higher concentrations. This hypersensitivity might be a result of the lower expression of cccA gene (encoding a vacuolar iron transporter which participates in iron detoxification) in the metRΔ mutant, especially under sulphur starvation conditions.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3757043&req=5

ppat-1003573-g008: Regulatory cross-talk between sulphur and iron metabolism.(A) Fungal cultures were shifted after prolonged growth in medium containing methionine as sole source of sulphur [Met (22 h)] to medium depleted for this amino acid but containing sulphate, and samples for RNA preparation were taken after the indicated time points. Northern hybridisation expression analysis of several genes directly related with iron metabolism was carried out. Although there is sufficient iron in the medium (100 µM), after eight hours of incubation in media containing sulphate (constituting sulphur starving conditions for the mutant) increased transcription of genes typically expressed under iron-depleted conditions (mirB: siderophore transporter; hapX: transcriptional activator of the iron regulon; sidA: L-ornithine monooxygenase; amcA: mitochondrial ornithine exporter) is observed in the metRΔ strain as well as decreased expression of genes whose products participate in iron consuming processes (cycA: cytochrome C; acoA: aconitase; isa1: mitochondrial cluster biosynthetic protein). (B) Relative quantity of ferricrocin (FC) in the respective fungal mycelia. FC levels are already elevated before the shift, and they increase nearly fivefold under sulphur starvation conditions. (C) The wild-type strain is able to grow in the presence of 10 mM of iron, a substantially high concentration that already provokes toxic effects. When sufficient methionine (5 mM) is present, the metRΔ mutant behaves as the wild-type and the reconstituted strain. However, under sulphur starvation, the mutant is not able grow on 0.5 mM of iron or higher concentrations. This hypersensitivity might be a result of the lower expression of cccA gene (encoding a vacuolar iron transporter which participates in iron detoxification) in the metRΔ mutant, especially under sulphur starvation conditions.
Mentions: It is well established that defects in mitochondrial Fe-S cluster biogenesis or transport induce transcription of the iron regulon [58], [59] and that Fe-S cluster-containing proteins participate directly in sensing iron availability in S. cerevisiae[60], [61]. Accordingly, we expected a regulatory cross-talk between sulphur assimilation and iron homeostasis in A. fumigatus, which was further indicated by the transcriptional profiling data (see above), however, which had not been tested in eukaryotes so far. We took advantage of our metRΔ mutant strain, which can be rapidly depleted for sulphur, to test this hypothesis. Overnight grown strains were shifted from culture media containing methionine to media depleted for this amino acid but containing sulphate. These media pose sulphur starving-conditions for the deletion strain but not for the wild-type or the reconstituted strain. After eight hours of incubation, although there was sufficient iron in the medium, the mutant strain increased transcription of several genes encoding proteins that participate in iron acquisition that are known to be upregulated under iron starvation, i.e. genes involved in siderophore biosynthesis (sidA), siderophore transport (mirB), mitochondrial ornithine export (amcA) and iron regulation (hapX) [57]. In contrast, the mutant decreased transcription of genes whose products participate in iron-consuming processes that are known to be downregulated under iron depleted conditions [40], i.e. genes encoding aconitase (acoA), cytochrome c (cycA) or components of the mitochondrial iron-sulfur-cluster biosynthetic machinery (isa1) (Fig. 8A). To analyse whether the cells were indeed depleted for iron, levels of iron chelated by ferricrocin (FC), the intracellular siderophore used for iron storage and transport [62], [63], were measured (Fig. 8B). Despite an expression pattern resembling that of iron starvation, the AfS167 mutant showed a nearly fivefold increased FC content. Combining ferricrocin analysis with total intracellular iron level measurements further underscored this imbalance in iron homeostasis (Table 4): MetR deficiency raises the cellular iron content 1.6-fold in the presence of methionine, which increases to 2.8-fold in its absence. The metRΔ mutant furthermore displays a 5-fold increased FC-chelated iron content under +Met conditions that is further enhanced to 7.5-fold when this S-sources is withdrawn. In conclusion, the cells indeed contain sufficient amounts of iron but display a defect in iron sensing and/or regulation. This dysregulation, causing an enhanced expression of iron uptake-related genes under sulphur starving conditions, translates into a phenotype of hypersensitivity to iron (Fig. 8C). At a low concentration of iron and methionine the metRΔ mutant was able to grow, although poorly due to the shortage of sulphur. This phenotype was recovered with higher availability of methionine. However, at higher iron concentrations, the wild-type strain could grow while the AfS167 mutant did not, unless a high amount of methionine was present in the medium. This might be the consequence of both the inability to shut down expression of iron uptake-related genes and of the lower cccA gene expression, encoding a recently described vacuolar transporter that has a prominent role in iron detoxification [64], in the metRΔ mutant especially under sulphur starvation (Fig. 8A), which results in iron accumulation (see Table 4: 2.4-fold increase for the metRΔ deletant under –Met conditions) to presumably toxic levels in the cytosol. Dysregulation of iron homeostasis was also tested in the wild-type strain by shifting its mycelium to a medium completely depleted for sulphur. However, expression of the iron regulon was not observed even after 24 hours of incubation (not shown), most likely because of the fact that the wild-type does not face such severe sulphur starving conditions as its metRΔ derivative apparently does.

Bottom Line: Surprisingly, A. fumigatus is able to utilize volatile sulphur compounds produced by its methionine catabolism, a process that has not been described before and that is MetR-dependent.The A. fumigatus MetR transcriptional activator is important for virulence in both leukopenic mice and an alternative mini-host model of aspergillosis, as it was essential for the development of pulmonary aspergillosis and supported the systemic dissemination of the fungus.Taken together, this study provides evidence that regulation of sulphur assimilation is not only crucial for A. fumigatus virulence but also affects the balance of iron in this prime opportunistic pathogen.

View Article: PubMed Central - PubMed

Affiliation: Research Center for Infectious Diseases, Julius-Maximilians-University Würzburg, Würzburg, Germany.

ABSTRACT
Sulphur is an essential element that all pathogens have to absorb from their surroundings in order to grow inside their infected host. Despite its importance, the relevance of sulphur assimilation in fungal virulence is largely unexplored. Here we report a role of the bZIP transcription factor MetR in sulphur assimilation and virulence of the human pathogen Aspergillus fumigatus. The MetR regulator is essential for growth on a variety of sulphur sources; remarkably, it is fundamental for assimilation of inorganic S-sources but dispensable for utilization of methionine. Accordingly, it strongly supports expression of genes directly related to inorganic sulphur assimilation but not of genes connected to methionine metabolism. On a broader scale, MetR orchestrates the comprehensive transcriptional adaptation to sulphur-starving conditions as demonstrated by digital gene expression analysis. Surprisingly, A. fumigatus is able to utilize volatile sulphur compounds produced by its methionine catabolism, a process that has not been described before and that is MetR-dependent. The A. fumigatus MetR transcriptional activator is important for virulence in both leukopenic mice and an alternative mini-host model of aspergillosis, as it was essential for the development of pulmonary aspergillosis and supported the systemic dissemination of the fungus. MetR action under sulphur-starving conditions is further required for proper iron regulation, which links regulation of sulphur metabolism to iron homeostasis and demonstrates an unprecedented regulatory crosstalk. Taken together, this study provides evidence that regulation of sulphur assimilation is not only crucial for A. fumigatus virulence but also affects the balance of iron in this prime opportunistic pathogen.

Show MeSH
Related in: MedlinePlus