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Identification of possible targets of the Aspergillus fumigatus CRZ1 homologue, CrzA.

Soriani FM, Malavazi I, Savoldi M, Espeso E, Dinamarco TM, Bernardes LA, Ferreira ME, Goldman MH, Goldman GH - BMC Microbiol. (2010)

Bottom Line: GFP::RcnA was mostly detected along the germling, did not accumulate in the nuclei and its location is not affected by the cellular response to calcium chloride.AfRcnA, one of these selected genes, encodes a modulator of calcineurin activity.A. nidulans AnRcnA mutation is suppressing CnaA mutation and it is responsible for modulating the calcineurin activity and mRNA accumulation of genes encoding calcium transporters.

View Article: PubMed Central - HTML - PubMed

Affiliation: Centro de Ciência e Tecnologia do Bioetanol and Faculdade de Ciências Farmacêuticas de Ribeirão Preto Universidade de São Paulo, São Paulo, Ribeirão Preto 14040-903, Brazil.

ABSTRACT

Background: Calcineurin, a serine/threonine-specific protein phosphatase, plays an important role in the control of cell morphology and virulence in fungi. Calcineurin regulates localization and activity of a transcription factor called CRZ1. Recently, we characterize Aspergillus fumigatus CRZ1 homologue, AfCrzA. Here, we investigate which pathways are influenced by A. fumigatus AfCrzA during a short pulse of calcium by comparatively determining the transcriptional profile of A. fumigatus wild type and DeltaAfcrzA mutant strains.

Results: We were able to observe 3,622 genes modulated in at least one timepoint in the mutant when compared to the wild type strain (3,211 and 411 at 10 and 30 minutes, respectively). Decreased mRNA abundance in the DeltacrzA was seen for genes encoding calcium transporters, transcription factors and genes that could be directly or indirectly involved in calcium metabolism. Increased mRNA accumulation was observed for some genes encoding proteins involved in stress response. AfCrzA overexpression in A. fumigatus increases the expression of several of these genes. The deleted strain of one of these genes, AfRcnA, belonging to a class of endogenous calcineurin regulators, calcipressins, had more calcineurin activity after exposure to calcium and was less sensitive to menadione 30 microM, hydrogen peroxide 2.5 mM, EGTA 25 mM, and MnCl2 25 mM. We constructed deletion, overexpression, and GFP fusion protein for the closely related A. nidulans AnRcnA. GFP::RcnA was mostly detected along the germling, did not accumulate in the nuclei and its location is not affected by the cellular response to calcium chloride.

Conclusion: We have performed a transcriptional profiling analysis of the A. fumigatus DeltaAfcrzA mutant strain exposed to calcium stress. This provided an excellent opportunity to identify genes and pathways that are under the influence of AfCrzA. AfRcnA, one of these selected genes, encodes a modulator of calcineurin activity. Concomitantly with A. fumigatus AfrcnA molecular analysis, we decided to exploit the conserved features of A. nidulans calcineurin system and investigated the A. nidulans AnRcnA homologue. A. nidulans AnRcnA mutation is suppressing CnaA mutation and it is responsible for modulating the calcineurin activity and mRNA accumulation of genes encoding calcium transporters.

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Overepression of A. fumigatus AfCrzA increased the mRNA accumulation of several genes. Fold increase in mRNA levels after the growth of the wild type and alcA::crzA mutant strain in MM+2% glycerol+2% ethanol for 6 hours at 37°C of (A) AfpmcB (Afu3g10690), (B) AfzfpA (Afu8g05010), (C) A. fumigatus Phospholipase D (Afu2g16520), (D) AfctfA (Afu4g03960), (E) Af BAR adaptor protein (Afu3g14230), (F) AfrcnA (Afu2g13060), (G) AfrfeF (Afu4g10200), (H) Af AAA ATPase (Afu4g04800), and (I) Afscf1 (Afu1g17370). The relative quantitation of all the genes and tubulin gene expression was determined by a standard curve (i.e., CT -values plotted against logarithm of the DNA copy number). The results are the means ± standard deviation of four sets of experiments. The values represent the number of times the genes are expressed compared to the corresponding wild type control strain (represented absolutely as 1.00).
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Figure 3: Overepression of A. fumigatus AfCrzA increased the mRNA accumulation of several genes. Fold increase in mRNA levels after the growth of the wild type and alcA::crzA mutant strain in MM+2% glycerol+2% ethanol for 6 hours at 37°C of (A) AfpmcB (Afu3g10690), (B) AfzfpA (Afu8g05010), (C) A. fumigatus Phospholipase D (Afu2g16520), (D) AfctfA (Afu4g03960), (E) Af BAR adaptor protein (Afu3g14230), (F) AfrcnA (Afu2g13060), (G) AfrfeF (Afu4g10200), (H) Af AAA ATPase (Afu4g04800), and (I) Afscf1 (Afu1g17370). The relative quantitation of all the genes and tubulin gene expression was determined by a standard curve (i.e., CT -values plotted against logarithm of the DNA copy number). The results are the means ± standard deviation of four sets of experiments. The values represent the number of times the genes are expressed compared to the corresponding wild type control strain (represented absolutely as 1.00).

Mentions: Overexpression of AfCrzA could reveal genes regulated by the calcineurin-AfCrzA pathway. Accordingly, we constructed an overexpression A. fumigatus AfCrzA strain by using the alcA promoter. The A. nidulans alcA promoter homologously replaced the AfcrzA promoter (for details of the construction, see Methods section). The alcA promoter is repressed by glucose, derepressed by glycerol and induced to high levels by ethanol or L-threonine [32]. When A. fumigatus is grown on glycerol 2% supplemented either with ethanol 2% or threonine 100 mM, AfcrzA mRNA accumulation in increased about 3.8- and 3.6-times, respectively compared to growth on glucose 4% (Figure 2A, left and right graphs). As expected, when the AfcrzA is repressed in the presence of glucose, the alcA::AfcrzA strain is more sensitive to calcium (Figure 2B); however, surprisingly high levels of AfCrzA mRNA accumulation also make the alcA::AfcrzA strain more calcium-sensitive (Figure 2B). These results suggest that CrzA overexpression could potentially disturb the mRNA accumulation of genes that are important for the calcium homeostasis in the cell, thus disturbing the calcium metabolism into the cell and consequently the growth in the presence of increasing calcium concentrations. To test this hypothesis we used real-time RT-PCR to verify the mRNA levels of selected genes observed as having decreased mRNA and increased accumulation (see Additional file 1, Table S1). As it can be seen in Figure 3, all tested genes to different extent have increased mRNA accumulation when the AfcrzA is overexpressed. The Af AAA ATPase (Afu4g04800) and AfScf1 (Afu1g17370) have increased mRNA accumulation when the ΔAfcrzA was exposed to calcium suggesting they are repressed by AfCrzA (see Figures 1E-F). We expected their mRNA accumulation would be reduced when AfCrzA is overexpressed. However, the mRNA levels of these genes were lower in the alcA::AfcrzA than in the ΔAfcrzA mutant strain (compare Figures 1E-F with Figures 3I-J), what could indicate that AfCrzA is partially controlling the mRNA accumulation levels of these genes. The genes encoding AfpmcB (Afu3g10690), AfrcnA (Afu2g13060), AfrfeF (Afu4g10200), and AfBAR adaptor protein (Afu3g14230) are about 14, 16, 13, and 250 times more expressed in the alcA::AfcrzA mutant than the wild type strain, respectively (Figure 3B and 3F-H). The AfpmcB (Afu3g10690) gene is A. fumigatus homologue of the yeast PMC1, a vacuolar Ca+2 ATPase involved in depleting cytosol of Ca+2 ions and preventing growth inhibition by activation of calcineurin in the presence of elevated concentrations of calcium [33]. The increased expression of this gene suggests AfCrzA is controlling directly or indirectly its expression. Furthermore, the increased mRNA accumulation of these calcium transporter-encoding genes is quite consistent taking into consideration the dramatic stress condition caused by the sudden increase in calcium concentration that needs either to be removed from the cytoplasm or transported to vacuoles. Considering the growth inhibition of the Aspergilli alcA::AfcrzA strain under high-Ca+2 conditions (see Figures 2A-B), one possible interpretation of these results is that the AfCrzA overexpression can inhibit the function of another factor that is necessary for growth under high-Ca+2 conditions.


Identification of possible targets of the Aspergillus fumigatus CRZ1 homologue, CrzA.

Soriani FM, Malavazi I, Savoldi M, Espeso E, Dinamarco TM, Bernardes LA, Ferreira ME, Goldman MH, Goldman GH - BMC Microbiol. (2010)

Overepression of A. fumigatus AfCrzA increased the mRNA accumulation of several genes. Fold increase in mRNA levels after the growth of the wild type and alcA::crzA mutant strain in MM+2% glycerol+2% ethanol for 6 hours at 37°C of (A) AfpmcB (Afu3g10690), (B) AfzfpA (Afu8g05010), (C) A. fumigatus Phospholipase D (Afu2g16520), (D) AfctfA (Afu4g03960), (E) Af BAR adaptor protein (Afu3g14230), (F) AfrcnA (Afu2g13060), (G) AfrfeF (Afu4g10200), (H) Af AAA ATPase (Afu4g04800), and (I) Afscf1 (Afu1g17370). The relative quantitation of all the genes and tubulin gene expression was determined by a standard curve (i.e., CT -values plotted against logarithm of the DNA copy number). The results are the means ± standard deviation of four sets of experiments. The values represent the number of times the genes are expressed compared to the corresponding wild type control strain (represented absolutely as 1.00).
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Related In: Results  -  Collection

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Show All Figures
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Figure 3: Overepression of A. fumigatus AfCrzA increased the mRNA accumulation of several genes. Fold increase in mRNA levels after the growth of the wild type and alcA::crzA mutant strain in MM+2% glycerol+2% ethanol for 6 hours at 37°C of (A) AfpmcB (Afu3g10690), (B) AfzfpA (Afu8g05010), (C) A. fumigatus Phospholipase D (Afu2g16520), (D) AfctfA (Afu4g03960), (E) Af BAR adaptor protein (Afu3g14230), (F) AfrcnA (Afu2g13060), (G) AfrfeF (Afu4g10200), (H) Af AAA ATPase (Afu4g04800), and (I) Afscf1 (Afu1g17370). The relative quantitation of all the genes and tubulin gene expression was determined by a standard curve (i.e., CT -values plotted against logarithm of the DNA copy number). The results are the means ± standard deviation of four sets of experiments. The values represent the number of times the genes are expressed compared to the corresponding wild type control strain (represented absolutely as 1.00).
Mentions: Overexpression of AfCrzA could reveal genes regulated by the calcineurin-AfCrzA pathway. Accordingly, we constructed an overexpression A. fumigatus AfCrzA strain by using the alcA promoter. The A. nidulans alcA promoter homologously replaced the AfcrzA promoter (for details of the construction, see Methods section). The alcA promoter is repressed by glucose, derepressed by glycerol and induced to high levels by ethanol or L-threonine [32]. When A. fumigatus is grown on glycerol 2% supplemented either with ethanol 2% or threonine 100 mM, AfcrzA mRNA accumulation in increased about 3.8- and 3.6-times, respectively compared to growth on glucose 4% (Figure 2A, left and right graphs). As expected, when the AfcrzA is repressed in the presence of glucose, the alcA::AfcrzA strain is more sensitive to calcium (Figure 2B); however, surprisingly high levels of AfCrzA mRNA accumulation also make the alcA::AfcrzA strain more calcium-sensitive (Figure 2B). These results suggest that CrzA overexpression could potentially disturb the mRNA accumulation of genes that are important for the calcium homeostasis in the cell, thus disturbing the calcium metabolism into the cell and consequently the growth in the presence of increasing calcium concentrations. To test this hypothesis we used real-time RT-PCR to verify the mRNA levels of selected genes observed as having decreased mRNA and increased accumulation (see Additional file 1, Table S1). As it can be seen in Figure 3, all tested genes to different extent have increased mRNA accumulation when the AfcrzA is overexpressed. The Af AAA ATPase (Afu4g04800) and AfScf1 (Afu1g17370) have increased mRNA accumulation when the ΔAfcrzA was exposed to calcium suggesting they are repressed by AfCrzA (see Figures 1E-F). We expected their mRNA accumulation would be reduced when AfCrzA is overexpressed. However, the mRNA levels of these genes were lower in the alcA::AfcrzA than in the ΔAfcrzA mutant strain (compare Figures 1E-F with Figures 3I-J), what could indicate that AfCrzA is partially controlling the mRNA accumulation levels of these genes. The genes encoding AfpmcB (Afu3g10690), AfrcnA (Afu2g13060), AfrfeF (Afu4g10200), and AfBAR adaptor protein (Afu3g14230) are about 14, 16, 13, and 250 times more expressed in the alcA::AfcrzA mutant than the wild type strain, respectively (Figure 3B and 3F-H). The AfpmcB (Afu3g10690) gene is A. fumigatus homologue of the yeast PMC1, a vacuolar Ca+2 ATPase involved in depleting cytosol of Ca+2 ions and preventing growth inhibition by activation of calcineurin in the presence of elevated concentrations of calcium [33]. The increased expression of this gene suggests AfCrzA is controlling directly or indirectly its expression. Furthermore, the increased mRNA accumulation of these calcium transporter-encoding genes is quite consistent taking into consideration the dramatic stress condition caused by the sudden increase in calcium concentration that needs either to be removed from the cytoplasm or transported to vacuoles. Considering the growth inhibition of the Aspergilli alcA::AfcrzA strain under high-Ca+2 conditions (see Figures 2A-B), one possible interpretation of these results is that the AfCrzA overexpression can inhibit the function of another factor that is necessary for growth under high-Ca+2 conditions.

Bottom Line: GFP::RcnA was mostly detected along the germling, did not accumulate in the nuclei and its location is not affected by the cellular response to calcium chloride.AfRcnA, one of these selected genes, encodes a modulator of calcineurin activity.A. nidulans AnRcnA mutation is suppressing CnaA mutation and it is responsible for modulating the calcineurin activity and mRNA accumulation of genes encoding calcium transporters.

View Article: PubMed Central - HTML - PubMed

Affiliation: Centro de Ciência e Tecnologia do Bioetanol and Faculdade de Ciências Farmacêuticas de Ribeirão Preto Universidade de São Paulo, São Paulo, Ribeirão Preto 14040-903, Brazil.

ABSTRACT

Background: Calcineurin, a serine/threonine-specific protein phosphatase, plays an important role in the control of cell morphology and virulence in fungi. Calcineurin regulates localization and activity of a transcription factor called CRZ1. Recently, we characterize Aspergillus fumigatus CRZ1 homologue, AfCrzA. Here, we investigate which pathways are influenced by A. fumigatus AfCrzA during a short pulse of calcium by comparatively determining the transcriptional profile of A. fumigatus wild type and DeltaAfcrzA mutant strains.

Results: We were able to observe 3,622 genes modulated in at least one timepoint in the mutant when compared to the wild type strain (3,211 and 411 at 10 and 30 minutes, respectively). Decreased mRNA abundance in the DeltacrzA was seen for genes encoding calcium transporters, transcription factors and genes that could be directly or indirectly involved in calcium metabolism. Increased mRNA accumulation was observed for some genes encoding proteins involved in stress response. AfCrzA overexpression in A. fumigatus increases the expression of several of these genes. The deleted strain of one of these genes, AfRcnA, belonging to a class of endogenous calcineurin regulators, calcipressins, had more calcineurin activity after exposure to calcium and was less sensitive to menadione 30 microM, hydrogen peroxide 2.5 mM, EGTA 25 mM, and MnCl2 25 mM. We constructed deletion, overexpression, and GFP fusion protein for the closely related A. nidulans AnRcnA. GFP::RcnA was mostly detected along the germling, did not accumulate in the nuclei and its location is not affected by the cellular response to calcium chloride.

Conclusion: We have performed a transcriptional profiling analysis of the A. fumigatus DeltaAfcrzA mutant strain exposed to calcium stress. This provided an excellent opportunity to identify genes and pathways that are under the influence of AfCrzA. AfRcnA, one of these selected genes, encodes a modulator of calcineurin activity. Concomitantly with A. fumigatus AfrcnA molecular analysis, we decided to exploit the conserved features of A. nidulans calcineurin system and investigated the A. nidulans AnRcnA homologue. A. nidulans AnRcnA mutation is suppressing CnaA mutation and it is responsible for modulating the calcineurin activity and mRNA accumulation of genes encoding calcium transporters.

Show MeSH
Related in: MedlinePlus