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Fungal Morphology, Iron Homeostasis, and Lipid Metabolism Regulated by a GATA Transcription Factor in Blastomyces dermatitidis.

Marty AJ, Broman AT, Zarnowski R, Dwyer TG, Bond LM, Lounes-Hadj Sahraoui A, Fontaine J, Ntambi JM, Keleş S, Kendziorski C, Gauthier GM - PLoS Pathog. (2015)

Bottom Line: This included genes involved with siderophore biosynthesis and uptake, iron homeostasis, and genes unrelated to iron assimilation.Chromatin immunoprecipitation, RNA interference, and overexpression analyses suggested that SREB was in a negative regulatory circuit with the bZIP transcription factor encoded by HAPX.Both SREB and HAPX affected morphogenesis at 22°C; however, large changes in transcript abundance by gene deletion for SREB or strong overexpression for HAPX were required to alter the phase transition.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of Wisconsin, Madison, Madison, Wisconsin, United States of America.

ABSTRACT
In response to temperature, Blastomyces dermatitidis converts between yeast and mold forms. Knowledge of the mechanism(s) underlying this response to temperature remains limited. In B. dermatitidis, we identified a GATA transcription factor, SREB, important for the transition to mold. Null mutants (SREBΔ) fail to fully complete the conversion to mold and cannot properly regulate siderophore biosynthesis. To capture the transcriptional response regulated by SREB early in the phase transition (0-48 hours), gene expression microarrays were used to compare SREB∆ to an isogenic wild type isolate. Analysis of the time course microarray data demonstrated SREB functioned as a transcriptional regulator at 37°C and 22°C. Bioinformatic and biochemical analyses indicated SREB was involved in diverse biological processes including iron homeostasis, biosynthesis of triacylglycerol and ergosterol, and lipid droplet formation. Integration of microarray data, bioinformatics, and chromatin immunoprecipitation identified a subset of genes directly bound and regulated by SREB in vivo in yeast (37°C) and during the phase transition to mold (22°C). This included genes involved with siderophore biosynthesis and uptake, iron homeostasis, and genes unrelated to iron assimilation. Functional analysis suggested that lipid droplets were actively metabolized during the phase transition and lipid metabolism may contribute to filamentous growth at 22°C. Chromatin immunoprecipitation, RNA interference, and overexpression analyses suggested that SREB was in a negative regulatory circuit with the bZIP transcription factor encoded by HAPX. Both SREB and HAPX affected morphogenesis at 22°C; however, large changes in transcript abundance by gene deletion for SREB or strong overexpression for HAPX were required to alter the phase transition.

No MeSH data available.


Related in: MedlinePlus

Genes in the SREB regulon involved with iron acquisition and homeostasis.(A) Heat map of genes in the SREB regulon involved with siderophore biosynthesis (orange text for the siderophore biosynthetic gene cluster and red text for ferricrocin biosynthetic genes), siderophore transport (green text), and ferric reduction (blue text). (B) Schematic of putative siderophore biosynthetic gene cluster. Locus number (BDBG #) for genes in the Blastomyces genome database at the Broad Institute (www.broadinstitute.org) is listed below the coding region of each gene. (C) Quantitative real-time PCR analysis of yeast (37°C) for a subset of genes in the SREB regulon involved with iron acquisition and homeostasis. The results were averaged from 2 experiments. (D) Reverse-phase high-pressure liquid chromatography analysis for ferricrocin, an intracellular storage siderophore, in wild-type and SREB∆ yeast.
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ppat.1004959.g004: Genes in the SREB regulon involved with iron acquisition and homeostasis.(A) Heat map of genes in the SREB regulon involved with siderophore biosynthesis (orange text for the siderophore biosynthetic gene cluster and red text for ferricrocin biosynthetic genes), siderophore transport (green text), and ferric reduction (blue text). (B) Schematic of putative siderophore biosynthetic gene cluster. Locus number (BDBG #) for genes in the Blastomyces genome database at the Broad Institute (www.broadinstitute.org) is listed below the coding region of each gene. (C) Quantitative real-time PCR analysis of yeast (37°C) for a subset of genes in the SREB regulon involved with iron acquisition and homeostasis. The results were averaged from 2 experiments. (D) Reverse-phase high-pressure liquid chromatography analysis for ferricrocin, an intracellular storage siderophore, in wild-type and SREB∆ yeast.

Mentions: Gene expression microarray analysis indicated SREB functioned as a major regulator of iron assimilation. SREB∆ failed to repress genes involved with siderophore biosynthesis and transport under iron-replete conditions at 37°C and 22°C (Fig 4A–4C). Moreover, a siderophore biosynthetic gene cluster (SBGC) containing 10 genes across 31.295 kb was identified (Fig 4B). With the exception of a gene predicted to encode a conserved hypothetical protein with unknown function (BDBG_00049), all genes in the predicted SBGC were differentially expressed in SREB∆ when compared to a WT isolate (Fig 4A–4C). DELTA-BLAST analysis against the NCBI (National Center for Biotechnology Information) database predicted that genes in the SBGC were involved with the biosynthesis of siderophores (dimerum acid and coprogen) secreted into the extracellular environment. Quantitative real-time PCR confirmed derepression of DE genes in the SBGC under iron-replete conditions in SREB∆ (Fig 4C). Two genes located outside the SBGC, BDBG_09503, BDBG_08208, were predicted to biosynthesize ferricrocin, an intracellular storage siderophore. Transcript abundance for BDBG_09503, which encodes a siderophore biosynthetic protein, was similar in SREB∆ and WT (Fig 4A and 4C). In contrast, transcript for a non-ribosomal siderophore biosynthesis peptide synthase SIDC (BDBG_08208) was 1.4 and 2.5-fold higher in SREB∆ than WT by microarray and qRT-PCR analyses, respectively (Fig 4A and 4C). Reverse-phase HPLC demonstrated elevated concentration of intracellular ferricrocin in SREB∆ compared to WT under iron-replete conditions (Fig 4D).


Fungal Morphology, Iron Homeostasis, and Lipid Metabolism Regulated by a GATA Transcription Factor in Blastomyces dermatitidis.

Marty AJ, Broman AT, Zarnowski R, Dwyer TG, Bond LM, Lounes-Hadj Sahraoui A, Fontaine J, Ntambi JM, Keleş S, Kendziorski C, Gauthier GM - PLoS Pathog. (2015)

Genes in the SREB regulon involved with iron acquisition and homeostasis.(A) Heat map of genes in the SREB regulon involved with siderophore biosynthesis (orange text for the siderophore biosynthetic gene cluster and red text for ferricrocin biosynthetic genes), siderophore transport (green text), and ferric reduction (blue text). (B) Schematic of putative siderophore biosynthetic gene cluster. Locus number (BDBG #) for genes in the Blastomyces genome database at the Broad Institute (www.broadinstitute.org) is listed below the coding region of each gene. (C) Quantitative real-time PCR analysis of yeast (37°C) for a subset of genes in the SREB regulon involved with iron acquisition and homeostasis. The results were averaged from 2 experiments. (D) Reverse-phase high-pressure liquid chromatography analysis for ferricrocin, an intracellular storage siderophore, in wild-type and SREB∆ yeast.
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Related In: Results  -  Collection

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Show All Figures
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ppat.1004959.g004: Genes in the SREB regulon involved with iron acquisition and homeostasis.(A) Heat map of genes in the SREB regulon involved with siderophore biosynthesis (orange text for the siderophore biosynthetic gene cluster and red text for ferricrocin biosynthetic genes), siderophore transport (green text), and ferric reduction (blue text). (B) Schematic of putative siderophore biosynthetic gene cluster. Locus number (BDBG #) for genes in the Blastomyces genome database at the Broad Institute (www.broadinstitute.org) is listed below the coding region of each gene. (C) Quantitative real-time PCR analysis of yeast (37°C) for a subset of genes in the SREB regulon involved with iron acquisition and homeostasis. The results were averaged from 2 experiments. (D) Reverse-phase high-pressure liquid chromatography analysis for ferricrocin, an intracellular storage siderophore, in wild-type and SREB∆ yeast.
Mentions: Gene expression microarray analysis indicated SREB functioned as a major regulator of iron assimilation. SREB∆ failed to repress genes involved with siderophore biosynthesis and transport under iron-replete conditions at 37°C and 22°C (Fig 4A–4C). Moreover, a siderophore biosynthetic gene cluster (SBGC) containing 10 genes across 31.295 kb was identified (Fig 4B). With the exception of a gene predicted to encode a conserved hypothetical protein with unknown function (BDBG_00049), all genes in the predicted SBGC were differentially expressed in SREB∆ when compared to a WT isolate (Fig 4A–4C). DELTA-BLAST analysis against the NCBI (National Center for Biotechnology Information) database predicted that genes in the SBGC were involved with the biosynthesis of siderophores (dimerum acid and coprogen) secreted into the extracellular environment. Quantitative real-time PCR confirmed derepression of DE genes in the SBGC under iron-replete conditions in SREB∆ (Fig 4C). Two genes located outside the SBGC, BDBG_09503, BDBG_08208, were predicted to biosynthesize ferricrocin, an intracellular storage siderophore. Transcript abundance for BDBG_09503, which encodes a siderophore biosynthetic protein, was similar in SREB∆ and WT (Fig 4A and 4C). In contrast, transcript for a non-ribosomal siderophore biosynthesis peptide synthase SIDC (BDBG_08208) was 1.4 and 2.5-fold higher in SREB∆ than WT by microarray and qRT-PCR analyses, respectively (Fig 4A and 4C). Reverse-phase HPLC demonstrated elevated concentration of intracellular ferricrocin in SREB∆ compared to WT under iron-replete conditions (Fig 4D).

Bottom Line: This included genes involved with siderophore biosynthesis and uptake, iron homeostasis, and genes unrelated to iron assimilation.Chromatin immunoprecipitation, RNA interference, and overexpression analyses suggested that SREB was in a negative regulatory circuit with the bZIP transcription factor encoded by HAPX.Both SREB and HAPX affected morphogenesis at 22°C; however, large changes in transcript abundance by gene deletion for SREB or strong overexpression for HAPX were required to alter the phase transition.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of Wisconsin, Madison, Madison, Wisconsin, United States of America.

ABSTRACT
In response to temperature, Blastomyces dermatitidis converts between yeast and mold forms. Knowledge of the mechanism(s) underlying this response to temperature remains limited. In B. dermatitidis, we identified a GATA transcription factor, SREB, important for the transition to mold. Null mutants (SREBΔ) fail to fully complete the conversion to mold and cannot properly regulate siderophore biosynthesis. To capture the transcriptional response regulated by SREB early in the phase transition (0-48 hours), gene expression microarrays were used to compare SREB∆ to an isogenic wild type isolate. Analysis of the time course microarray data demonstrated SREB functioned as a transcriptional regulator at 37°C and 22°C. Bioinformatic and biochemical analyses indicated SREB was involved in diverse biological processes including iron homeostasis, biosynthesis of triacylglycerol and ergosterol, and lipid droplet formation. Integration of microarray data, bioinformatics, and chromatin immunoprecipitation identified a subset of genes directly bound and regulated by SREB in vivo in yeast (37°C) and during the phase transition to mold (22°C). This included genes involved with siderophore biosynthesis and uptake, iron homeostasis, and genes unrelated to iron assimilation. Functional analysis suggested that lipid droplets were actively metabolized during the phase transition and lipid metabolism may contribute to filamentous growth at 22°C. Chromatin immunoprecipitation, RNA interference, and overexpression analyses suggested that SREB was in a negative regulatory circuit with the bZIP transcription factor encoded by HAPX. Both SREB and HAPX affected morphogenesis at 22°C; however, large changes in transcript abundance by gene deletion for SREB or strong overexpression for HAPX were required to alter the phase transition.

No MeSH data available.


Related in: MedlinePlus