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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.


Fatty acid supplementation for wild type and SREB∆.(A) Percentage of WT and SREB∆ with yeast morphology, germ tube development, and hyphae at 22°C for cells grown in media supplemented with 0.5 mM palmitic acid (16:0) or 0.5 mM stearic acid (18:0). Controls included DMSO only and cells grown in media without DMSO or saturated fatty acid (untreated). At least 200 cells were counted in duplicate. Results were averaged from 2 independent experiments. (B) Percentage of filaments that contained 0–4 or 5 LDs per 10 μm segment at 24 and 48-hrs 22°C for WT and SREB∆ cells (untreated, DMSO only, 16:0 and 18:0). LDs were quantified per 10 μm segment along the length of filaments from at least 30 cells. Results were averaged from 2 independent experiments. (C) BODIPY 493/503 staining of lipid droplets at 24 and 48-hrs 22°C for WT and SREB∆ cells (untreated, DMSO only, 16:0 and 18:0). Scale bar equals 10 μm.
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ppat.1004959.g008: Fatty acid supplementation for wild type and SREB∆.(A) Percentage of WT and SREB∆ with yeast morphology, germ tube development, and hyphae at 22°C for cells grown in media supplemented with 0.5 mM palmitic acid (16:0) or 0.5 mM stearic acid (18:0). Controls included DMSO only and cells grown in media without DMSO or saturated fatty acid (untreated). At least 200 cells were counted in duplicate. Results were averaged from 2 independent experiments. (B) Percentage of filaments that contained 0–4 or 5 LDs per 10 μm segment at 24 and 48-hrs 22°C for WT and SREB∆ cells (untreated, DMSO only, 16:0 and 18:0). LDs were quantified per 10 μm segment along the length of filaments from at least 30 cells. Results were averaged from 2 independent experiments. (C) BODIPY 493/503 staining of lipid droplets at 24 and 48-hrs 22°C for WT and SREB∆ cells (untreated, DMSO only, 16:0 and 18:0). Scale bar equals 10 μm.

Mentions: In eukaryotic cells, fatty acids (FAs) serve as an important substrate for TAG and sterol ester biosynthesis [44,45]. To investigate if exogenous fatty acids can influence the phase transition or LD formation, SREB∆ cells were treated with saturated (16:0, 18:0) and unsaturated (16:1n7, 18:1n9) fatty acids. Experiments with exogenous TAG were not performed because fungal cells are unable to uptake this glycerolipid. The poor solubility of saturated FAs in aqueous solutions necessitated dissolving 16:0 and 18:0 in DMSO prior to supplementation of iron-replete HMM. Treatment of SREB∆ with 0.5 mM palmitic (16:0) or 0.5 mM stearic (18:0) acid accelerated the morphologic switch at 22°C compared to control strains (SREB∆ untreated, SREB∆ DMSO) without affecting the phase transition of WT cells (untreated, DMSO, 16:0, 18:0) (Fig 8A). At 24-hrs 22°C, exogenous 16:0 and 18:0 increased germ tube formation in SREB∆ versus SREB∆ untreated and SREB∆ DMSO (Fig 8A). At 24 and 48-hrs 22°C, SREB∆ treated with 16:0 or 18:0 exhibited increased conversion to hyphae and decreased number of yeast cells compared to controls (SREB∆ untreated, SREB∆ DMSO) (Fig 8A). The morphology of yeast cells at 37°C was unaffected by 16:0 or 18:0 (S7 Fig). Although DMSO did not affect the phase transition (Fig 8A), higher concentrations of DMSO needed to solubilize ≥ 1 mM 16:0 or 18:0 hindered the morphologic switch to mold for WT and SREB∆. BODIPY staining demonstrated that exogenous 16:0 and 18:0 restored LDs in the growing filaments of SREB∆ at 24-hrs 22°C (Fig 8B and 8C). Median LD per 10 μm increased from 1 for SREB∆ controls (untreated, DMSO) to 5 for SREB∆ 16:0 and 2.5 for SREB∆ 18:0. Moreover, the percentage of 10 μm filament segments with 5 LDs increased 4-fold for SREB∆ 16:0 and 2.2-fold for SREB∆ 18:0 compared to controls (SREB∆ untreated, SREB∆ DMSO) (Fig 8B). The increase in LDs was transient and limited to the 24-hr 22°C time point (Fig 8A–8C). At 48-hrs 22°C, the median number of LDs per 10 μm for SREB∆ 16:0 and 18:0 declined to 2 and the percentage of 10 μm filament segments with ≤ 4 LDs increased (Fig 8A–8C). Exogenous 16:0 or 18:0 did not affect the number of lipid droplets for SREB∆ at 37°C and 6-hrs 22°C (Fig 8B and 8C, and S7 Fig). WT cells treated with 16:0 or 18:0 had similar number of LDs as controls (median 6–7 LD per 10 μm filament segment at 24 and 48-hrs 22°C) (Fig 8B and 8C). SREB∆ cells treated with 0.5 mM oleic acid (18:1n9) exhibited similar morphologic and LD defects as untreated SREB∆ cells at 48-hrs 22°C (S8 Fig). Treatment of cells with 0.5 mM palmitoleic acid (16:1n7) was lethal for WT and SREB∆; reducing 16:1n7 concentrations to 0.250 mM or 0.125 mM did not improve cell viability (S8 Fig).


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)

Fatty acid supplementation for wild type and SREB∆.(A) Percentage of WT and SREB∆ with yeast morphology, germ tube development, and hyphae at 22°C for cells grown in media supplemented with 0.5 mM palmitic acid (16:0) or 0.5 mM stearic acid (18:0). Controls included DMSO only and cells grown in media without DMSO or saturated fatty acid (untreated). At least 200 cells were counted in duplicate. Results were averaged from 2 independent experiments. (B) Percentage of filaments that contained 0–4 or 5 LDs per 10 μm segment at 24 and 48-hrs 22°C for WT and SREB∆ cells (untreated, DMSO only, 16:0 and 18:0). LDs were quantified per 10 μm segment along the length of filaments from at least 30 cells. Results were averaged from 2 independent experiments. (C) BODIPY 493/503 staining of lipid droplets at 24 and 48-hrs 22°C for WT and SREB∆ cells (untreated, DMSO only, 16:0 and 18:0). Scale bar equals 10 μm.
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Related In: Results  -  Collection

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ppat.1004959.g008: Fatty acid supplementation for wild type and SREB∆.(A) Percentage of WT and SREB∆ with yeast morphology, germ tube development, and hyphae at 22°C for cells grown in media supplemented with 0.5 mM palmitic acid (16:0) or 0.5 mM stearic acid (18:0). Controls included DMSO only and cells grown in media without DMSO or saturated fatty acid (untreated). At least 200 cells were counted in duplicate. Results were averaged from 2 independent experiments. (B) Percentage of filaments that contained 0–4 or 5 LDs per 10 μm segment at 24 and 48-hrs 22°C for WT and SREB∆ cells (untreated, DMSO only, 16:0 and 18:0). LDs were quantified per 10 μm segment along the length of filaments from at least 30 cells. Results were averaged from 2 independent experiments. (C) BODIPY 493/503 staining of lipid droplets at 24 and 48-hrs 22°C for WT and SREB∆ cells (untreated, DMSO only, 16:0 and 18:0). Scale bar equals 10 μm.
Mentions: In eukaryotic cells, fatty acids (FAs) serve as an important substrate for TAG and sterol ester biosynthesis [44,45]. To investigate if exogenous fatty acids can influence the phase transition or LD formation, SREB∆ cells were treated with saturated (16:0, 18:0) and unsaturated (16:1n7, 18:1n9) fatty acids. Experiments with exogenous TAG were not performed because fungal cells are unable to uptake this glycerolipid. The poor solubility of saturated FAs in aqueous solutions necessitated dissolving 16:0 and 18:0 in DMSO prior to supplementation of iron-replete HMM. Treatment of SREB∆ with 0.5 mM palmitic (16:0) or 0.5 mM stearic (18:0) acid accelerated the morphologic switch at 22°C compared to control strains (SREB∆ untreated, SREB∆ DMSO) without affecting the phase transition of WT cells (untreated, DMSO, 16:0, 18:0) (Fig 8A). At 24-hrs 22°C, exogenous 16:0 and 18:0 increased germ tube formation in SREB∆ versus SREB∆ untreated and SREB∆ DMSO (Fig 8A). At 24 and 48-hrs 22°C, SREB∆ treated with 16:0 or 18:0 exhibited increased conversion to hyphae and decreased number of yeast cells compared to controls (SREB∆ untreated, SREB∆ DMSO) (Fig 8A). The morphology of yeast cells at 37°C was unaffected by 16:0 or 18:0 (S7 Fig). Although DMSO did not affect the phase transition (Fig 8A), higher concentrations of DMSO needed to solubilize ≥ 1 mM 16:0 or 18:0 hindered the morphologic switch to mold for WT and SREB∆. BODIPY staining demonstrated that exogenous 16:0 and 18:0 restored LDs in the growing filaments of SREB∆ at 24-hrs 22°C (Fig 8B and 8C). Median LD per 10 μm increased from 1 for SREB∆ controls (untreated, DMSO) to 5 for SREB∆ 16:0 and 2.5 for SREB∆ 18:0. Moreover, the percentage of 10 μm filament segments with 5 LDs increased 4-fold for SREB∆ 16:0 and 2.2-fold for SREB∆ 18:0 compared to controls (SREB∆ untreated, SREB∆ DMSO) (Fig 8B). The increase in LDs was transient and limited to the 24-hr 22°C time point (Fig 8A–8C). At 48-hrs 22°C, the median number of LDs per 10 μm for SREB∆ 16:0 and 18:0 declined to 2 and the percentage of 10 μm filament segments with ≤ 4 LDs increased (Fig 8A–8C). Exogenous 16:0 or 18:0 did not affect the number of lipid droplets for SREB∆ at 37°C and 6-hrs 22°C (Fig 8B and 8C, and S7 Fig). WT cells treated with 16:0 or 18:0 had similar number of LDs as controls (median 6–7 LD per 10 μm filament segment at 24 and 48-hrs 22°C) (Fig 8B and 8C). SREB∆ cells treated with 0.5 mM oleic acid (18:1n9) exhibited similar morphologic and LD defects as untreated SREB∆ cells at 48-hrs 22°C (S8 Fig). Treatment of cells with 0.5 mM palmitoleic acid (16:1n7) was lethal for WT and SREB∆; reducing 16:1n7 concentrations to 0.250 mM or 0.125 mM did not improve cell viability (S8 Fig).

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.