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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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Phenotypic analysis and germination rate of an A. fumigatus strain deleted for its metR gene.(A) A metRΔ deletion mutant is unable to grow on any of the tested inorganic sulphur sources. Conidia of the indicated strains were inoculated on Aspergillus minimal medium supplemented with various sources of sulphur as well as complex media to monitor growth after three days of incubation at 37°C. Among the tested organic sources, the metRΔ strain grows only on methionine or homocysteine. When the media are depleted for nitrogen and sulphur simultaneously, cysteine and glutathione can be exploited as sources for both elements. The metRΔ strain also grows poorly on a substrate prepared from a porcine lung (PLA) unless supplemented with methionine. In all conditions, the phenotype of the metR+ revertant strain was indistinguishable from the wild-type progenitor. (B) From conidia inoculated in liquid culture, rates of germination were deduced in dependency of the S-source. Germ tube formation of the metRΔ strain is slightly delayed with respect to the wild-type in the presence of 5 mM methionine as S-source, while in the presence of 2 mM sulphate, the mutant is not able to germinate within 11 hours of incubation.
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ppat-1003573-g002: Phenotypic analysis and germination rate of an A. fumigatus strain deleted for its metR gene.(A) A metRΔ deletion mutant is unable to grow on any of the tested inorganic sulphur sources. Conidia of the indicated strains were inoculated on Aspergillus minimal medium supplemented with various sources of sulphur as well as complex media to monitor growth after three days of incubation at 37°C. Among the tested organic sources, the metRΔ strain grows only on methionine or homocysteine. When the media are depleted for nitrogen and sulphur simultaneously, cysteine and glutathione can be exploited as sources for both elements. The metRΔ strain also grows poorly on a substrate prepared from a porcine lung (PLA) unless supplemented with methionine. In all conditions, the phenotype of the metR+ revertant strain was indistinguishable from the wild-type progenitor. (B) From conidia inoculated in liquid culture, rates of germination were deduced in dependency of the S-source. Germ tube formation of the metRΔ strain is slightly delayed with respect to the wild-type in the presence of 5 mM methionine as S-source, while in the presence of 2 mM sulphate, the mutant is not able to germinate within 11 hours of incubation.

Mentions: To gain insights into the cellular function of the metR gene product, a full deletion strain of A. fumigatus was constructed by homologous gene replacement employing a self-excising recyclable cassette that contains a hygromycin B resistance gene as selectable marker [46], [47]. Southern analysis of the resulting strains AfS166 [metR::six-β-rec/hygroR-six] and AfS167 [metR::six] confirmed the homologous replacement and the excising event, respectively (Fig. S1A). A preliminary phenotypic analysis revealed that a metR deletant is unable to grow in the presence of sulphate as sole source of sulphur (Fig. 2A). This allowed us to reintroduce the metR gene at its original locus without using any selection marker but the presence of sulphate (SO42−) as the only source of sulphur. In order to differentiate between the desired reconstituted strain and its wild-type progenitor, a silent mutation was introduced in the gene's coding sequence to create an additional BstEII restriction site. Southern analysis confirmed the correct integration event for a representative isolate (Fig. S1B).


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)

Phenotypic analysis and germination rate of an A. fumigatus strain deleted for its metR gene.(A) A metRΔ deletion mutant is unable to grow on any of the tested inorganic sulphur sources. Conidia of the indicated strains were inoculated on Aspergillus minimal medium supplemented with various sources of sulphur as well as complex media to monitor growth after three days of incubation at 37°C. Among the tested organic sources, the metRΔ strain grows only on methionine or homocysteine. When the media are depleted for nitrogen and sulphur simultaneously, cysteine and glutathione can be exploited as sources for both elements. The metRΔ strain also grows poorly on a substrate prepared from a porcine lung (PLA) unless supplemented with methionine. In all conditions, the phenotype of the metR+ revertant strain was indistinguishable from the wild-type progenitor. (B) From conidia inoculated in liquid culture, rates of germination were deduced in dependency of the S-source. Germ tube formation of the metRΔ strain is slightly delayed with respect to the wild-type in the presence of 5 mM methionine as S-source, while in the presence of 2 mM sulphate, the mutant is not able to germinate within 11 hours of incubation.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1003573-g002: Phenotypic analysis and germination rate of an A. fumigatus strain deleted for its metR gene.(A) A metRΔ deletion mutant is unable to grow on any of the tested inorganic sulphur sources. Conidia of the indicated strains were inoculated on Aspergillus minimal medium supplemented with various sources of sulphur as well as complex media to monitor growth after three days of incubation at 37°C. Among the tested organic sources, the metRΔ strain grows only on methionine or homocysteine. When the media are depleted for nitrogen and sulphur simultaneously, cysteine and glutathione can be exploited as sources for both elements. The metRΔ strain also grows poorly on a substrate prepared from a porcine lung (PLA) unless supplemented with methionine. In all conditions, the phenotype of the metR+ revertant strain was indistinguishable from the wild-type progenitor. (B) From conidia inoculated in liquid culture, rates of germination were deduced in dependency of the S-source. Germ tube formation of the metRΔ strain is slightly delayed with respect to the wild-type in the presence of 5 mM methionine as S-source, while in the presence of 2 mM sulphate, the mutant is not able to germinate within 11 hours of incubation.
Mentions: To gain insights into the cellular function of the metR gene product, a full deletion strain of A. fumigatus was constructed by homologous gene replacement employing a self-excising recyclable cassette that contains a hygromycin B resistance gene as selectable marker [46], [47]. Southern analysis of the resulting strains AfS166 [metR::six-β-rec/hygroR-six] and AfS167 [metR::six] confirmed the homologous replacement and the excising event, respectively (Fig. S1A). A preliminary phenotypic analysis revealed that a metR deletant is unable to grow in the presence of sulphate as sole source of sulphur (Fig. 2A). This allowed us to reintroduce the metR gene at its original locus without using any selection marker but the presence of sulphate (SO42−) as the only source of sulphur. In order to differentiate between the desired reconstituted strain and its wild-type progenitor, a silent mutation was introduced in the gene's coding sequence to create an additional BstEII restriction site. Southern analysis confirmed the correct integration event for a representative isolate (Fig. S1B).

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