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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

Transcription analysis of sulphur-related genes in the presence of varying sulphur sources.Analysis of the transcriptional expression of several genes that participate in sulphur metabolism by Northern blot hybridisation. RNA samples had been isolated from the metRΔ deletant and its wild-type progenitor strain after shifting pre-grown fungal cultures to medium containing the indicated source of sulphur (Met, Cys, and SO42−) or lacking any S-Source (-S). Transcript steady-state levels were monitored from cultures after one hour of growth with the exception of the metAT transcript, for which culturing had been extended to eight hours additionally. For all blots, rRNAs served as loading control, autoradiographies are representatives from three independent, reproducible experimental replicates. (A) Expression of the metR gene itself is not regulated by the sulphur source. Expression of all genes from the sulphate assimilation pathway - sA, sB, sC, and sD - is elevated under sulphur starving conditions in a MetR-dependent manner. Expression of the arylsulphatase encoding gene completely depends on MetR. (B) Expression of the putative methionine transporter encoding genes (mupA and mupC) is constitutive. Transcription of the methionine synthase encoding gene (metH) is upregulated under sulphur-starving conditions, independently of MetR. Regulation of the expression of the methionine aminotransferase encoding gene (metAT) is MetR-independent. Expression of metAT is elevated in the presence of methionine. In addition, the metRΔ mutant increases metAT expression under sulphur-depleted conditions. This upregulation also occurred after eight hours incubation in the presence of sulphate. (C) Transcription of the cysteine synthase (cysB) is slightly upregulated with a sulphur source other than cysteine in a MetR dependent manner. Expression of the putative cysteine permease (cynA) is upregulated under sulphur-starving conditions in a MetR-dependent manner. (D) MetR binds to the promoter regions of selected candidate genes of the sulphate assimilation pathway, like sB, sD, sC, or AFUA_8G02520 but not the iron regulator gene hapX as demonstrated by chromatin immunoprecipitation analyses. Shown are inverse images from agarose gel electrophoreses after semi-quantitative PCRs on fixed and sheared chromatin samples enriched from the MetR-GFP strain AfS171 in comparison to samples from the untagged wild-type control strain ATCC 46645; − and + specify negative (without template) and positive (genomic DNA as template) controls, respectively, while figures indicate the numbers of PCR cycles and M stands for the DNA size standard.
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ppat-1003573-g005: Transcription analysis of sulphur-related genes in the presence of varying sulphur sources.Analysis of the transcriptional expression of several genes that participate in sulphur metabolism by Northern blot hybridisation. RNA samples had been isolated from the metRΔ deletant and its wild-type progenitor strain after shifting pre-grown fungal cultures to medium containing the indicated source of sulphur (Met, Cys, and SO42−) or lacking any S-Source (-S). Transcript steady-state levels were monitored from cultures after one hour of growth with the exception of the metAT transcript, for which culturing had been extended to eight hours additionally. For all blots, rRNAs served as loading control, autoradiographies are representatives from three independent, reproducible experimental replicates. (A) Expression of the metR gene itself is not regulated by the sulphur source. Expression of all genes from the sulphate assimilation pathway - sA, sB, sC, and sD - is elevated under sulphur starving conditions in a MetR-dependent manner. Expression of the arylsulphatase encoding gene completely depends on MetR. (B) Expression of the putative methionine transporter encoding genes (mupA and mupC) is constitutive. Transcription of the methionine synthase encoding gene (metH) is upregulated under sulphur-starving conditions, independently of MetR. Regulation of the expression of the methionine aminotransferase encoding gene (metAT) is MetR-independent. Expression of metAT is elevated in the presence of methionine. In addition, the metRΔ mutant increases metAT expression under sulphur-depleted conditions. This upregulation also occurred after eight hours incubation in the presence of sulphate. (C) Transcription of the cysteine synthase (cysB) is slightly upregulated with a sulphur source other than cysteine in a MetR dependent manner. Expression of the putative cysteine permease (cynA) is upregulated under sulphur-starving conditions in a MetR-dependent manner. (D) MetR binds to the promoter regions of selected candidate genes of the sulphate assimilation pathway, like sB, sD, sC, or AFUA_8G02520 but not the iron regulator gene hapX as demonstrated by chromatin immunoprecipitation analyses. Shown are inverse images from agarose gel electrophoreses after semi-quantitative PCRs on fixed and sheared chromatin samples enriched from the MetR-GFP strain AfS171 in comparison to samples from the untagged wild-type control strain ATCC 46645; − and + specify negative (without template) and positive (genomic DNA as template) controls, respectively, while figures indicate the numbers of PCR cycles and M stands for the DNA size standard.

Mentions: Initially, transcript levels of the metR gene itself were checked to observe that its transcription is apparently not regulated by the nature of the sulphur source (Fig. 5A), resembling the situation in A. nidulans[32] but contrasting findings in N. crassa[24], [25]. Interestingly, a second hybridising signal was detected for the metR transcript under S-starvation conditions, indicating alternative processing of the encoding transcript. In order to further understand why the metRΔ mutant is unable to grow on oxidized inorganic sulphur sources, expression of all genes encoding enzymes of the sulphate assimilation pathway, which are sulphate permease (sB), ATP-sulphurylase (sC), APS-kinase (sD), PAPS-reductase (sA) and sulphite reductase, as well as expression of one arylsulphatase-encoding gene (required for utilization of sulphur esters, i.e. nitrophenyl sulphate) was checked (Fig. 5A). Upregulation of the sulphate permease-, ATP-sulphurylase- and APS-kinase-encoding genes under sulphur-starving conditions depended on the presence of MetR factor. For the sB gene, a second, longer transcript became evident under sulphur starvation. Transcription levels of the genes coding for PAPS reductase and sulphite reductase were decreased in the absence of the MetR factor on all S-sources in comparison to the wild-type. Furthermore, expression of the arylsulphatase-encoding gene was completely shut down in the mutant. This transcriptional pattern agrees with and partially explains the incapacity of the metRΔ deletant to grow on inorganic sulphur sources.


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)

Transcription analysis of sulphur-related genes in the presence of varying sulphur sources.Analysis of the transcriptional expression of several genes that participate in sulphur metabolism by Northern blot hybridisation. RNA samples had been isolated from the metRΔ deletant and its wild-type progenitor strain after shifting pre-grown fungal cultures to medium containing the indicated source of sulphur (Met, Cys, and SO42−) or lacking any S-Source (-S). Transcript steady-state levels were monitored from cultures after one hour of growth with the exception of the metAT transcript, for which culturing had been extended to eight hours additionally. For all blots, rRNAs served as loading control, autoradiographies are representatives from three independent, reproducible experimental replicates. (A) Expression of the metR gene itself is not regulated by the sulphur source. Expression of all genes from the sulphate assimilation pathway - sA, sB, sC, and sD - is elevated under sulphur starving conditions in a MetR-dependent manner. Expression of the arylsulphatase encoding gene completely depends on MetR. (B) Expression of the putative methionine transporter encoding genes (mupA and mupC) is constitutive. Transcription of the methionine synthase encoding gene (metH) is upregulated under sulphur-starving conditions, independently of MetR. Regulation of the expression of the methionine aminotransferase encoding gene (metAT) is MetR-independent. Expression of metAT is elevated in the presence of methionine. In addition, the metRΔ mutant increases metAT expression under sulphur-depleted conditions. This upregulation also occurred after eight hours incubation in the presence of sulphate. (C) Transcription of the cysteine synthase (cysB) is slightly upregulated with a sulphur source other than cysteine in a MetR dependent manner. Expression of the putative cysteine permease (cynA) is upregulated under sulphur-starving conditions in a MetR-dependent manner. (D) MetR binds to the promoter regions of selected candidate genes of the sulphate assimilation pathway, like sB, sD, sC, or AFUA_8G02520 but not the iron regulator gene hapX as demonstrated by chromatin immunoprecipitation analyses. Shown are inverse images from agarose gel electrophoreses after semi-quantitative PCRs on fixed and sheared chromatin samples enriched from the MetR-GFP strain AfS171 in comparison to samples from the untagged wild-type control strain ATCC 46645; − and + specify negative (without template) and positive (genomic DNA as template) controls, respectively, while figures indicate the numbers of PCR cycles and M stands for the DNA size standard.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1003573-g005: Transcription analysis of sulphur-related genes in the presence of varying sulphur sources.Analysis of the transcriptional expression of several genes that participate in sulphur metabolism by Northern blot hybridisation. RNA samples had been isolated from the metRΔ deletant and its wild-type progenitor strain after shifting pre-grown fungal cultures to medium containing the indicated source of sulphur (Met, Cys, and SO42−) or lacking any S-Source (-S). Transcript steady-state levels were monitored from cultures after one hour of growth with the exception of the metAT transcript, for which culturing had been extended to eight hours additionally. For all blots, rRNAs served as loading control, autoradiographies are representatives from three independent, reproducible experimental replicates. (A) Expression of the metR gene itself is not regulated by the sulphur source. Expression of all genes from the sulphate assimilation pathway - sA, sB, sC, and sD - is elevated under sulphur starving conditions in a MetR-dependent manner. Expression of the arylsulphatase encoding gene completely depends on MetR. (B) Expression of the putative methionine transporter encoding genes (mupA and mupC) is constitutive. Transcription of the methionine synthase encoding gene (metH) is upregulated under sulphur-starving conditions, independently of MetR. Regulation of the expression of the methionine aminotransferase encoding gene (metAT) is MetR-independent. Expression of metAT is elevated in the presence of methionine. In addition, the metRΔ mutant increases metAT expression under sulphur-depleted conditions. This upregulation also occurred after eight hours incubation in the presence of sulphate. (C) Transcription of the cysteine synthase (cysB) is slightly upregulated with a sulphur source other than cysteine in a MetR dependent manner. Expression of the putative cysteine permease (cynA) is upregulated under sulphur-starving conditions in a MetR-dependent manner. (D) MetR binds to the promoter regions of selected candidate genes of the sulphate assimilation pathway, like sB, sD, sC, or AFUA_8G02520 but not the iron regulator gene hapX as demonstrated by chromatin immunoprecipitation analyses. Shown are inverse images from agarose gel electrophoreses after semi-quantitative PCRs on fixed and sheared chromatin samples enriched from the MetR-GFP strain AfS171 in comparison to samples from the untagged wild-type control strain ATCC 46645; − and + specify negative (without template) and positive (genomic DNA as template) controls, respectively, while figures indicate the numbers of PCR cycles and M stands for the DNA size standard.
Mentions: Initially, transcript levels of the metR gene itself were checked to observe that its transcription is apparently not regulated by the nature of the sulphur source (Fig. 5A), resembling the situation in A. nidulans[32] but contrasting findings in N. crassa[24], [25]. Interestingly, a second hybridising signal was detected for the metR transcript under S-starvation conditions, indicating alternative processing of the encoding transcript. In order to further understand why the metRΔ mutant is unable to grow on oxidized inorganic sulphur sources, expression of all genes encoding enzymes of the sulphate assimilation pathway, which are sulphate permease (sB), ATP-sulphurylase (sC), APS-kinase (sD), PAPS-reductase (sA) and sulphite reductase, as well as expression of one arylsulphatase-encoding gene (required for utilization of sulphur esters, i.e. nitrophenyl sulphate) was checked (Fig. 5A). Upregulation of the sulphate permease-, ATP-sulphurylase- and APS-kinase-encoding genes under sulphur-starving conditions depended on the presence of MetR factor. For the sB gene, a second, longer transcript became evident under sulphur starvation. Transcription levels of the genes coding for PAPS reductase and sulphite reductase were decreased in the absence of the MetR factor on all S-sources in comparison to the wild-type. Furthermore, expression of the arylsulphatase-encoding gene was completely shut down in the mutant. This transcriptional pattern agrees with and partially explains the incapacity of the metRΔ deletant to grow on inorganic sulphur sources.

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