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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.


Lipid droplets in wild type and SREB∆.(A) BODIPY 493/503 was used to stain for lipid droplets (LD) in wild type (WT) and SREB∆ at 37°C and 6, 24, and 48-hrs following a drop in temperature to 22°C. Corresponding bright field microscopic images (light) are below the fluorescent images. Scale bar is 10 μm (B) Median number of lipid droplets per yeast, yeast cell body, and 10 μm filament were quantified at 37°C and 22°C (range is in parentheses). To adjust for differences in filamentous growth at 24 and 48-hrs 22°C, LDs were quantified per 10 μm segment along the length of the filament. LDs were quantified from 50 cells in duplicate. (C) Percentage of filaments that contained 0–4 or ≥ 5 LDs per 10 μm segment at 24 and 48-hrs 22°C.
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ppat.1004959.g007: Lipid droplets in wild type and SREB∆.(A) BODIPY 493/503 was used to stain for lipid droplets (LD) in wild type (WT) and SREB∆ at 37°C and 6, 24, and 48-hrs following a drop in temperature to 22°C. Corresponding bright field microscopic images (light) are below the fluorescent images. Scale bar is 10 μm (B) Median number of lipid droplets per yeast, yeast cell body, and 10 μm filament were quantified at 37°C and 22°C (range is in parentheses). To adjust for differences in filamentous growth at 24 and 48-hrs 22°C, LDs were quantified per 10 μm segment along the length of the filament. LDs were quantified from 50 cells in duplicate. (C) Percentage of filaments that contained 0–4 or ≥ 5 LDs per 10 μm segment at 24 and 48-hrs 22°C.

Mentions: The reduction in TAG and ergosterol in SREB∆ prompted investigation for alteration in lipid droplets at 37°C and 22°C. Lipid droplets are organelles that consist of a neutral lipid core of TAG and sterol esters surrounded by a phospholipid monolayer intercalated with proteins [41]. Sterol esters are derived from ergosterol and intermediates in the ergosterol biosynthetic pathway [42]. SREB∆ was analyzed for defective lipid droplet formation using BODIPY, a fluorescent dye that is specific for lipid droplets (LDs) [43]. LDs were quantified in cells with yeast morphology at 37°C and 6-hrs 22°C. At 24 and 48-hrs 22°C, LDs in the yeast cell body as well as the emerging filament (germ tube or hyphae) were quantified. To adjust for the differences in filament length for SREB∆ compared to WT, LDs were quantified per 10 μm segments along the length of the filament. At 37°C and 6-hrs 22°C, there were no differences in LD abundance in yeast cells between SREB∆ and WT (Fig 7A and 7B). At 24-hrs 22°C, the median LD abundance was sharply reduced in SREB∆ filaments versus WT (0 versus 7); however, the median number of LD per yeast cell body for SREB∆ was similar to WT (16.5 versus 18) (Fig 7A and 7B). At 48-hrs 22°C, the median number of LDs per 10 μm segment (0 versus 6) and yeast cell body (10 versus 17) was decreased in SREB∆ compared to WT (Fig 7A and 7B). At 24 and 48-hrs 22°C, more than 80% of 10 μm segments analyzed for SREB∆ had ≤ 4 LD, whereas for WT, less than 33% of 10 μm segments had 4 or fewer LD (Fig 7C).


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)

Lipid droplets in wild type and SREB∆.(A) BODIPY 493/503 was used to stain for lipid droplets (LD) in wild type (WT) and SREB∆ at 37°C and 6, 24, and 48-hrs following a drop in temperature to 22°C. Corresponding bright field microscopic images (light) are below the fluorescent images. Scale bar is 10 μm (B) Median number of lipid droplets per yeast, yeast cell body, and 10 μm filament were quantified at 37°C and 22°C (range is in parentheses). To adjust for differences in filamentous growth at 24 and 48-hrs 22°C, LDs were quantified per 10 μm segment along the length of the filament. LDs were quantified from 50 cells in duplicate. (C) Percentage of filaments that contained 0–4 or ≥ 5 LDs per 10 μm segment at 24 and 48-hrs 22°C.
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ppat.1004959.g007: Lipid droplets in wild type and SREB∆.(A) BODIPY 493/503 was used to stain for lipid droplets (LD) in wild type (WT) and SREB∆ at 37°C and 6, 24, and 48-hrs following a drop in temperature to 22°C. Corresponding bright field microscopic images (light) are below the fluorescent images. Scale bar is 10 μm (B) Median number of lipid droplets per yeast, yeast cell body, and 10 μm filament were quantified at 37°C and 22°C (range is in parentheses). To adjust for differences in filamentous growth at 24 and 48-hrs 22°C, LDs were quantified per 10 μm segment along the length of the filament. LDs were quantified from 50 cells in duplicate. (C) Percentage of filaments that contained 0–4 or ≥ 5 LDs per 10 μm segment at 24 and 48-hrs 22°C.
Mentions: The reduction in TAG and ergosterol in SREB∆ prompted investigation for alteration in lipid droplets at 37°C and 22°C. Lipid droplets are organelles that consist of a neutral lipid core of TAG and sterol esters surrounded by a phospholipid monolayer intercalated with proteins [41]. Sterol esters are derived from ergosterol and intermediates in the ergosterol biosynthetic pathway [42]. SREB∆ was analyzed for defective lipid droplet formation using BODIPY, a fluorescent dye that is specific for lipid droplets (LDs) [43]. LDs were quantified in cells with yeast morphology at 37°C and 6-hrs 22°C. At 24 and 48-hrs 22°C, LDs in the yeast cell body as well as the emerging filament (germ tube or hyphae) were quantified. To adjust for the differences in filament length for SREB∆ compared to WT, LDs were quantified per 10 μm segments along the length of the filament. At 37°C and 6-hrs 22°C, there were no differences in LD abundance in yeast cells between SREB∆ and WT (Fig 7A and 7B). At 24-hrs 22°C, the median LD abundance was sharply reduced in SREB∆ filaments versus WT (0 versus 7); however, the median number of LD per yeast cell body for SREB∆ was similar to WT (16.5 versus 18) (Fig 7A and 7B). At 48-hrs 22°C, the median number of LDs per 10 μm segment (0 versus 6) and yeast cell body (10 versus 17) was decreased in SREB∆ compared to WT (Fig 7A and 7B). At 24 and 48-hrs 22°C, more than 80% of 10 μm segments analyzed for SREB∆ had ≤ 4 LD, whereas for WT, less than 33% of 10 μm segments had 4 or fewer LD (Fig 7C).

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.