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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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AfRcnA affects the mRNA accumulation of genes whose expression is influenced by AfcrzA. Fold increase in mRNA levels after the incubation ot he wild type and ΔAfrcnA mutant strains with 200 mM CaCl2 for 10 and 30 minutes of (A) AfrfeF (Afu4g10200), (B) Af Bar adaptor protein (Afu3g14230), (C) A. fumigatus phospholipase D (Afu2g16520), (D) Af AAA ATPase (Afu4g04800), and (E) 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 5: AfRcnA affects the mRNA accumulation of genes whose expression is influenced by AfcrzA. Fold increase in mRNA levels after the incubation ot he wild type and ΔAfrcnA mutant strains with 200 mM CaCl2 for 10 and 30 minutes of (A) AfrfeF (Afu4g10200), (B) Af Bar adaptor protein (Afu3g14230), (C) A. fumigatus phospholipase D (Afu2g16520), (D) Af AAA ATPase (Afu4g04800), and (E) 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: We also investigated how the AfrcnA deletion would affect the mRNA accumulation of the genes observed as modulated by AfCrzA (see Figure 1). Thus, we exposed both the wild type and AfΔrcnA mutant strains to CaCl2 200 mM for 10 and 30 minutes and measured the mRNA accumulation of these genes (Figure 5). The absence of AfRcnA has a very heterogeneous influence on the mRNA accumulation of these genes. The AfrfeF (Afu4g10200) and Af AAA ATPase (Afu4g04800) genes have increased mRNA accumulation in the absence of AfrcnA when compared to the wild type strain (about 1.5- and 5.0-times and about the same and 5-times increased at 10 and 30 minutes, respectively; Figures 5A and 5D). In contrast, the A. fumigatus phospholipase D (Afu2g16520) gene has lower mRNA accumulation of 3.6- and 5.0-times in the AfΔrcnA mutant than the wild type strain (Figure 5C). The mRNA accumulation of the Af BAR (Afu3g14230) and AfScf1 (Afu1g17370) genes is not affected by the absence of AfrcnA (Figures 5B and 5E). These data emphasize the complex influence of AfRcnA on the calcineurin pathway, both stimulating and inhibiting genes in this pathway.


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)

AfRcnA affects the mRNA accumulation of genes whose expression is influenced by AfcrzA. Fold increase in mRNA levels after the incubation ot he wild type and ΔAfrcnA mutant strains with 200 mM CaCl2 for 10 and 30 minutes of (A) AfrfeF (Afu4g10200), (B) Af Bar adaptor protein (Afu3g14230), (C) A. fumigatus phospholipase D (Afu2g16520), (D) Af AAA ATPase (Afu4g04800), and (E) 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).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2818617&req=5

Figure 5: AfRcnA affects the mRNA accumulation of genes whose expression is influenced by AfcrzA. Fold increase in mRNA levels after the incubation ot he wild type and ΔAfrcnA mutant strains with 200 mM CaCl2 for 10 and 30 minutes of (A) AfrfeF (Afu4g10200), (B) Af Bar adaptor protein (Afu3g14230), (C) A. fumigatus phospholipase D (Afu2g16520), (D) Af AAA ATPase (Afu4g04800), and (E) 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: We also investigated how the AfrcnA deletion would affect the mRNA accumulation of the genes observed as modulated by AfCrzA (see Figure 1). Thus, we exposed both the wild type and AfΔrcnA mutant strains to CaCl2 200 mM for 10 and 30 minutes and measured the mRNA accumulation of these genes (Figure 5). The absence of AfRcnA has a very heterogeneous influence on the mRNA accumulation of these genes. The AfrfeF (Afu4g10200) and Af AAA ATPase (Afu4g04800) genes have increased mRNA accumulation in the absence of AfrcnA when compared to the wild type strain (about 1.5- and 5.0-times and about the same and 5-times increased at 10 and 30 minutes, respectively; Figures 5A and 5D). In contrast, the A. fumigatus phospholipase D (Afu2g16520) gene has lower mRNA accumulation of 3.6- and 5.0-times in the AfΔrcnA mutant than the wild type strain (Figure 5C). The mRNA accumulation of the Af BAR (Afu3g14230) and AfScf1 (Afu1g17370) genes is not affected by the absence of AfrcnA (Figures 5B and 5E). These data emphasize the complex influence of AfRcnA on the calcineurin pathway, both stimulating and inhibiting genes in this pathway.

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