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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.


HAPX overexpression affects the phase transition in B. dermatitidis.(A) Similar to SREB∆, HAPX overexpression (OE) strains #8, #18, and #22 grew as yeast at 37°C, but failed to complete conversion to mold following a downshift in temperature to 22°C. In contrast, wild-type (WT) and empty vector (EV) strains converted to mold at 22°C. Scale bar is 10 μm. (B) Quantitative RT-PCR analysis of HAPX transcript abundance in controls (WT, EV) and overexpression (OE# 8, OE#18, OE#22) strains. HAPX OE strains had 15.5–53.1-fold increased HAPX transcript abundance compared to controls. (C) Quantitative RT-PCR demonstrated a 3.4–5.8-fold decrease in SREB transcript abundance in OE strains #8 and #18 compared to WT and EV controls. SREB transcript abundance in OE #22 exhibited a 0.9–1.4-fold change compared to controls (WT, EV). All strains were grown in liquid HMM supplemented with 10 μM FeSO4. Quantitative RT-PCR results were averaged from 2 experiments.
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ppat.1004959.g010: HAPX overexpression affects the phase transition in B. dermatitidis.(A) Similar to SREB∆, HAPX overexpression (OE) strains #8, #18, and #22 grew as yeast at 37°C, but failed to complete conversion to mold following a downshift in temperature to 22°C. In contrast, wild-type (WT) and empty vector (EV) strains converted to mold at 22°C. Scale bar is 10 μm. (B) Quantitative RT-PCR analysis of HAPX transcript abundance in controls (WT, EV) and overexpression (OE# 8, OE#18, OE#22) strains. HAPX OE strains had 15.5–53.1-fold increased HAPX transcript abundance compared to controls. (C) Quantitative RT-PCR demonstrated a 3.4–5.8-fold decrease in SREB transcript abundance in OE strains #8 and #18 compared to WT and EV controls. SREB transcript abundance in OE #22 exhibited a 0.9–1.4-fold change compared to controls (WT, EV). All strains were grown in liquid HMM supplemented with 10 μM FeSO4. Quantitative RT-PCR results were averaged from 2 experiments.

Mentions: Gene expression microarray data indicated that SREB altered the phase transition independent of VMA1 and HGRA; these genes are not DE in SREB∆. To test the importance of a subset of SREB-bound genes on the phase transition, we overexpressed WD and HAPX in wild-type strain 26199 under the control of an H2B promoter. WD was selected for overexpression because WD-repeat proteins can be involved with transcriptional regulation and affect fungal development [46,47]. HAPX is also involved with transcriptional regulation and in Aspergillus spp., it is in a negative regulatory circuit with SREA, a homolog of SREB [48,49]. These genes were overexpressed rather than silenced because they were derepressed in SREB∆. The H2B promoter was active at 37°C, 22°C, and during the phase transition when measured by qRT-PCR (Ct values 21 at 37°C and 22°C for H2B transcript). WD overexpression strains grew as yeast and converted normally to mold at 22°C. HAPX overexpression strains (OE 8, 18, 22) were morphologically similar to SREB∆ following a drop in temperature from 37°C to 22°C (Fig 10A). Quantitative RT-PCR analysis demonstrated 15.5–53.1-fold increase in HAPX transcript abundance in OE strains compared to WT (Fig 10B). SREB transcript abundance was decreased in OE 8 and OE 18, but was similar to WT and empty vector for OE 22 (Fig 10C). In HAPX OE 8 and 18, the reduction in SREB transcript was unstable and normalized to WT levels over serial passage; however, the morphologic defect and HAPX overexpression persisted. Unlike deletion of SREB, overexpression of HAPX did not adversely affect LD formation at 24 and 48-hrs 22°C.


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)

HAPX overexpression affects the phase transition in B. dermatitidis.(A) Similar to SREB∆, HAPX overexpression (OE) strains #8, #18, and #22 grew as yeast at 37°C, but failed to complete conversion to mold following a downshift in temperature to 22°C. In contrast, wild-type (WT) and empty vector (EV) strains converted to mold at 22°C. Scale bar is 10 μm. (B) Quantitative RT-PCR analysis of HAPX transcript abundance in controls (WT, EV) and overexpression (OE# 8, OE#18, OE#22) strains. HAPX OE strains had 15.5–53.1-fold increased HAPX transcript abundance compared to controls. (C) Quantitative RT-PCR demonstrated a 3.4–5.8-fold decrease in SREB transcript abundance in OE strains #8 and #18 compared to WT and EV controls. SREB transcript abundance in OE #22 exhibited a 0.9–1.4-fold change compared to controls (WT, EV). All strains were grown in liquid HMM supplemented with 10 μM FeSO4. Quantitative RT-PCR results were averaged from 2 experiments.
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ppat.1004959.g010: HAPX overexpression affects the phase transition in B. dermatitidis.(A) Similar to SREB∆, HAPX overexpression (OE) strains #8, #18, and #22 grew as yeast at 37°C, but failed to complete conversion to mold following a downshift in temperature to 22°C. In contrast, wild-type (WT) and empty vector (EV) strains converted to mold at 22°C. Scale bar is 10 μm. (B) Quantitative RT-PCR analysis of HAPX transcript abundance in controls (WT, EV) and overexpression (OE# 8, OE#18, OE#22) strains. HAPX OE strains had 15.5–53.1-fold increased HAPX transcript abundance compared to controls. (C) Quantitative RT-PCR demonstrated a 3.4–5.8-fold decrease in SREB transcript abundance in OE strains #8 and #18 compared to WT and EV controls. SREB transcript abundance in OE #22 exhibited a 0.9–1.4-fold change compared to controls (WT, EV). All strains were grown in liquid HMM supplemented with 10 μM FeSO4. Quantitative RT-PCR results were averaged from 2 experiments.
Mentions: Gene expression microarray data indicated that SREB altered the phase transition independent of VMA1 and HGRA; these genes are not DE in SREB∆. To test the importance of a subset of SREB-bound genes on the phase transition, we overexpressed WD and HAPX in wild-type strain 26199 under the control of an H2B promoter. WD was selected for overexpression because WD-repeat proteins can be involved with transcriptional regulation and affect fungal development [46,47]. HAPX is also involved with transcriptional regulation and in Aspergillus spp., it is in a negative regulatory circuit with SREA, a homolog of SREB [48,49]. These genes were overexpressed rather than silenced because they were derepressed in SREB∆. The H2B promoter was active at 37°C, 22°C, and during the phase transition when measured by qRT-PCR (Ct values 21 at 37°C and 22°C for H2B transcript). WD overexpression strains grew as yeast and converted normally to mold at 22°C. HAPX overexpression strains (OE 8, 18, 22) were morphologically similar to SREB∆ following a drop in temperature from 37°C to 22°C (Fig 10A). Quantitative RT-PCR analysis demonstrated 15.5–53.1-fold increase in HAPX transcript abundance in OE strains compared to WT (Fig 10B). SREB transcript abundance was decreased in OE 8 and OE 18, but was similar to WT and empty vector for OE 22 (Fig 10C). In HAPX OE 8 and 18, the reduction in SREB transcript was unstable and normalized to WT levels over serial passage; however, the morphologic defect and HAPX overexpression persisted. Unlike deletion of SREB, overexpression of HAPX did not adversely affect LD formation at 24 and 48-hrs 22°C.

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.