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Integrative analysis of the heat shock response in Aspergillus fumigatus.

Albrecht D, Guthke R, Brakhage AA, Kniemeyer O - BMC Genomics (2010)

Bottom Line: To improve 2D gel image analysis results, protein spot quantitation was optimized by missing value imputation and normalization.Differentially regulated proteins were compared to previously published transcriptome data of A. fumigatus.Until now, this factor has only been found in vertebrates.

View Article: PubMed Central - HTML - PubMed

Affiliation: Leibniz Institute for Natural Product Research and Infection Biology, Hans-Knöll-Institute, Jena, Germany.

ABSTRACT

Background: Aspergillus fumigatus is a thermotolerant human-pathogenic mold and the most common cause of invasive aspergillosis (IA) in immunocompromised patients. Its predominance is based on several factors most of which are still unknown. The thermotolerance of A. fumigatus is one of the traits which have been assigned to pathogenicity. It allows the fungus to grow at temperatures up to and above that of a fevered human host. To elucidate the mechanisms of heat resistance, we analyzed the change of the A. fumigatus proteome during a temperature shift from 30 degrees C to 48 degrees C by 2D-fluorescence difference gel electrophoresis (DIGE). To improve 2D gel image analysis results, protein spot quantitation was optimized by missing value imputation and normalization. Differentially regulated proteins were compared to previously published transcriptome data of A. fumigatus. The study was augmented by bioinformatical analysis of transcription factor binding sites (TFBSs) in the promoter region of genes whose corresponding proteins were differentially regulated upon heat shock.

Results: 91 differentially regulated protein spots, representing 64 different proteins, were identified by mass spectrometry (MS). They showed a continuous up-, down- or an oscillating regulation. Many of the identified proteins were involved in protein folding (chaperones), oxidative stress response, signal transduction, transcription, translation, carbohydrate and nitrogen metabolism. A correlation between alteration of transcript levels and corresponding proteins was detected for half of the differentially regulated proteins. Interestingly, some previously undescribed putative targets for the heat shock regulator Hsf1 were identified. This provides evidence for Hsf1-dependent regulation of mannitol biosynthesis, translation, cytoskeletal dynamics and cell division in A. fumigatus. Furthermore, computational analysis of promoters revealed putative binding sites for an AP-2alpha-like transcription factor upstream of some heat shock induced genes. Until now, this factor has only been found in vertebrates.

Conclusions: Our newly established DIGE data analysis workflow yields improved data quality and is widely applicable for other DIGE datasets. Our findings suggest that the heat shock response in A. fumigatus differs from already well-studied yeasts and other filamentous fungi.

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Heatmap of potentially Hsf1 regulated proteins and transcripts. Red color depicts upregulation, green color depicts downregulation. The gene/protein names are shown as well as the detected HSE.
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Figure 5: Heatmap of potentially Hsf1 regulated proteins and transcripts. Red color depicts upregulation, green color depicts downregulation. The gene/protein names are shown as well as the detected HSE.

Mentions: In yeast, heat shock is largely governed by the transcription factors Hsf1, Msn2/4 and Hac1. Many targets of those have already been elucidated [11,12,14,43,44]. Hsf1 was very recently found to be upregulated in A. fumigatus under heat shock [18]. We looked for putative Hsf1 binding signatures [12,14] in the genome of A. fumigatus. By using ScanProsite [45], 17 genes with a potential heat shock element (HSE) in their promoter region were detected (see Figure 4 for motif logos and Figure 5 for heatmap of transcript and protein regulation). Proteins probably regulated by Hsf1 include chaperones (HSP70, HSP78, mitochondrial HSP60, Sti1), enzymes of the oxidative stress response (cytochrome C peroxidase Ccp1, allergen Asp F3), signal transduction (protein phosphotase 2a 65 kd regulatory subunit, conserved lysine rich protein), carbohydrate and nitrogen metabolism (Hexokinase Kxk, mannitol-1-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase Gnd1, nitrite reductase NiiA, 3-isopropylmalate dehydrogenase), protein biosynthesis/translation (eukaryotic translation initiation factor 4, histidyl-tRNA synthetase, DNAJ domain protein Psi) and transcription (glycine-rich RNA-binding protein) (for explicit binding sites see additional file 4, sequence_analysis.xls). By using MEME [46], one additional chaperone (BiP/Kar2) and one transport protein (nuclear movement protein NudC) with slightly modified HSE motifs were found. Transcriptional activation of genes coding for chaperones is well known from yeast and higher organisms, but for some other genes (Ccp1, protein phosphotase 2a, conserves lysine rich protein, mannitol-1-phosphate dehydrogenase, Idp1, 3-isopropylmalae dehydrogenase, eukaryotic translation initiation factor 4, histidyl-tRNA synthetase, glycine-rich RNA-binding protein, NudC) regulation by Hsf1 has not been previously elucidated for yeast (for comparison with yeast homologues see additional file 4). For the transcription factors Msn2/4 and Hac1 neither ScanProsite nor MEME provided useful results, since the binding motifs are very short. In addition to the Hsf1 binding sites, MEME identified a possible binding motif for an AP-2alphaA-like transcription factor in 11 of the 64 differentially regulated proteins (AFUA_1G05610, AFUA_4G07710, AFUA_1G10130, AFUA_5G04170, AFUA_5G07340, AFUA_2G17110, AFUA_2G10660, AFUA_3G09320, AFUA_6G12660, AFUA_1G02030, AFUA_7G01860; see Figure 4 for motif logo). The AP-2 family of transcription factors regulates proliferation and differentiation during embryonic development in animals [47]. To date, no AP-2 homologues have been detected in any eukaryotic microorganism.


Integrative analysis of the heat shock response in Aspergillus fumigatus.

Albrecht D, Guthke R, Brakhage AA, Kniemeyer O - BMC Genomics (2010)

Heatmap of potentially Hsf1 regulated proteins and transcripts. Red color depicts upregulation, green color depicts downregulation. The gene/protein names are shown as well as the detected HSE.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2820008&req=5

Figure 5: Heatmap of potentially Hsf1 regulated proteins and transcripts. Red color depicts upregulation, green color depicts downregulation. The gene/protein names are shown as well as the detected HSE.
Mentions: In yeast, heat shock is largely governed by the transcription factors Hsf1, Msn2/4 and Hac1. Many targets of those have already been elucidated [11,12,14,43,44]. Hsf1 was very recently found to be upregulated in A. fumigatus under heat shock [18]. We looked for putative Hsf1 binding signatures [12,14] in the genome of A. fumigatus. By using ScanProsite [45], 17 genes with a potential heat shock element (HSE) in their promoter region were detected (see Figure 4 for motif logos and Figure 5 for heatmap of transcript and protein regulation). Proteins probably regulated by Hsf1 include chaperones (HSP70, HSP78, mitochondrial HSP60, Sti1), enzymes of the oxidative stress response (cytochrome C peroxidase Ccp1, allergen Asp F3), signal transduction (protein phosphotase 2a 65 kd regulatory subunit, conserved lysine rich protein), carbohydrate and nitrogen metabolism (Hexokinase Kxk, mannitol-1-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase Gnd1, nitrite reductase NiiA, 3-isopropylmalate dehydrogenase), protein biosynthesis/translation (eukaryotic translation initiation factor 4, histidyl-tRNA synthetase, DNAJ domain protein Psi) and transcription (glycine-rich RNA-binding protein) (for explicit binding sites see additional file 4, sequence_analysis.xls). By using MEME [46], one additional chaperone (BiP/Kar2) and one transport protein (nuclear movement protein NudC) with slightly modified HSE motifs were found. Transcriptional activation of genes coding for chaperones is well known from yeast and higher organisms, but for some other genes (Ccp1, protein phosphotase 2a, conserves lysine rich protein, mannitol-1-phosphate dehydrogenase, Idp1, 3-isopropylmalae dehydrogenase, eukaryotic translation initiation factor 4, histidyl-tRNA synthetase, glycine-rich RNA-binding protein, NudC) regulation by Hsf1 has not been previously elucidated for yeast (for comparison with yeast homologues see additional file 4). For the transcription factors Msn2/4 and Hac1 neither ScanProsite nor MEME provided useful results, since the binding motifs are very short. In addition to the Hsf1 binding sites, MEME identified a possible binding motif for an AP-2alphaA-like transcription factor in 11 of the 64 differentially regulated proteins (AFUA_1G05610, AFUA_4G07710, AFUA_1G10130, AFUA_5G04170, AFUA_5G07340, AFUA_2G17110, AFUA_2G10660, AFUA_3G09320, AFUA_6G12660, AFUA_1G02030, AFUA_7G01860; see Figure 4 for motif logo). The AP-2 family of transcription factors regulates proliferation and differentiation during embryonic development in animals [47]. To date, no AP-2 homologues have been detected in any eukaryotic microorganism.

Bottom Line: To improve 2D gel image analysis results, protein spot quantitation was optimized by missing value imputation and normalization.Differentially regulated proteins were compared to previously published transcriptome data of A. fumigatus.Until now, this factor has only been found in vertebrates.

View Article: PubMed Central - HTML - PubMed

Affiliation: Leibniz Institute for Natural Product Research and Infection Biology, Hans-Knöll-Institute, Jena, Germany.

ABSTRACT

Background: Aspergillus fumigatus is a thermotolerant human-pathogenic mold and the most common cause of invasive aspergillosis (IA) in immunocompromised patients. Its predominance is based on several factors most of which are still unknown. The thermotolerance of A. fumigatus is one of the traits which have been assigned to pathogenicity. It allows the fungus to grow at temperatures up to and above that of a fevered human host. To elucidate the mechanisms of heat resistance, we analyzed the change of the A. fumigatus proteome during a temperature shift from 30 degrees C to 48 degrees C by 2D-fluorescence difference gel electrophoresis (DIGE). To improve 2D gel image analysis results, protein spot quantitation was optimized by missing value imputation and normalization. Differentially regulated proteins were compared to previously published transcriptome data of A. fumigatus. The study was augmented by bioinformatical analysis of transcription factor binding sites (TFBSs) in the promoter region of genes whose corresponding proteins were differentially regulated upon heat shock.

Results: 91 differentially regulated protein spots, representing 64 different proteins, were identified by mass spectrometry (MS). They showed a continuous up-, down- or an oscillating regulation. Many of the identified proteins were involved in protein folding (chaperones), oxidative stress response, signal transduction, transcription, translation, carbohydrate and nitrogen metabolism. A correlation between alteration of transcript levels and corresponding proteins was detected for half of the differentially regulated proteins. Interestingly, some previously undescribed putative targets for the heat shock regulator Hsf1 were identified. This provides evidence for Hsf1-dependent regulation of mannitol biosynthesis, translation, cytoskeletal dynamics and cell division in A. fumigatus. Furthermore, computational analysis of promoters revealed putative binding sites for an AP-2alpha-like transcription factor upstream of some heat shock induced genes. Until now, this factor has only been found in vertebrates.

Conclusions: Our newly established DIGE data analysis workflow yields improved data quality and is widely applicable for other DIGE datasets. Our findings suggest that the heat shock response in A. fumigatus differs from already well-studied yeasts and other filamentous fungi.

Show MeSH
Related in: MedlinePlus