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Different Stress-Induced Calcium Signatures Are Reported by Aequorin-Mediated Calcium Measurements in Living Cells of Aspergillus fumigatus.

Muñoz A, Bertuzzi M, Bettgenhaeuser J, Iakobachvili N, Bignell EM, Read ND - PLoS ONE (2015)

Bottom Line: The 'signatures' of the transient [Ca2+]c responses to extracellular stimuli were found to be dose- and age-dependent.Moreover, Ca2+-signatures associated with each physico-chemical treatment were found to be unique, suggesting the involvement of heterogeneous combinations of Ca2+-signalling components in each stress response.The Ca2+-chelator BAPTA potently inhibited the initial responses to most stressors in accordance with a critical role for extracellular Ca2+ in initiating the stress responses.

View Article: PubMed Central - PubMed

Affiliation: Manchester Fungal Infection Group, Institute of Inflammation and Repair, University of Manchester, Manchester, United Kingdom; Fungal Cell Biology Group, Institute of Cell Biology, University of Edinburgh, Edinburgh, United Kingdom.

ABSTRACT
Aspergillus fumigatus is an inhaled fungal pathogen of human lungs, the developmental growth of which is reliant upon Ca2+-mediated signalling. Ca2+ signalling has regulatory significance in all eukaryotic cells but how A. fumigatus uses intracellular Ca2+ signals to respond to stresses imposed by the mammalian lung is poorly understood. In this work, A. fumigatus strains derived from the clinical isolate CEA10, and a non-homologous recombination mutant ΔakuBKU80, were engineered to express the bioluminescent Ca2+-reporter aequorin. An aequorin-mediated method for routine Ca2+ measurements during the early stages of colony initiation was successfully developed and dynamic changes in cytosolic free calcium ([Ca2+]c) in response to extracellular stimuli were measured. The response to extracellular challenges (hypo- and hyper-osmotic shock, mechanical perturbation, high extracellular Ca2+, oxidative stress or exposure to human serum) that the fungus might be exposed to during infection, were analysed in living conidial germlings. The 'signatures' of the transient [Ca2+]c responses to extracellular stimuli were found to be dose- and age-dependent. Moreover, Ca2+-signatures associated with each physico-chemical treatment were found to be unique, suggesting the involvement of heterogeneous combinations of Ca2+-signalling components in each stress response. Concordant with the involvement of Ca2+-calmodulin complexes in these Ca2+-mediated responses, the calmodulin inhibitor trifluoperazine (TFP) induced changes in the Ca2+-signatures to all the challenges. The Ca2+-chelator BAPTA potently inhibited the initial responses to most stressors in accordance with a critical role for extracellular Ca2+ in initiating the stress responses.

No MeSH data available.


Related in: MedlinePlus

Pretreatment with the Ca2+-chelator BAPTA or the calmodulin inhibitor TFP differentially impacts upon Ca2+-signalling and homeostasis during A. fumigatus responses to stressors.After growth for 20.5 h at 25°C, A. fumigatus AEQCEA10 cultures were pre-treated for 30 min with either 5 mM BAPTA or 50 μM TFP prior to challenge with stressors. [Ca2+]c amplitudes (measured simultaneously to stress application) and post-stimulatory [Ca2+]c resting levels (10 min after stress) are represented as [Ca2+]c values (A-B) and as fold change (Log2 ratios of treated/untreated) with the modulators BAPTA (C-D) or TFP (E-F) under the different stress conditions indicated. For clarity, average values ± SD of the [Ca2+]c amplitudes and post-stimulatory [Ca2+]c resting levels are shown; however full Ca2+-signatures using the pretreatment with these modulators are plotted in S9 Fig Statistical analysis was performed using a 1-way ANOVA. * p < 0.05, ** p < 0.01, *** p < 0.005, **** p < 0.001.
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pone.0138008.g004: Pretreatment with the Ca2+-chelator BAPTA or the calmodulin inhibitor TFP differentially impacts upon Ca2+-signalling and homeostasis during A. fumigatus responses to stressors.After growth for 20.5 h at 25°C, A. fumigatus AEQCEA10 cultures were pre-treated for 30 min with either 5 mM BAPTA or 50 μM TFP prior to challenge with stressors. [Ca2+]c amplitudes (measured simultaneously to stress application) and post-stimulatory [Ca2+]c resting levels (10 min after stress) are represented as [Ca2+]c values (A-B) and as fold change (Log2 ratios of treated/untreated) with the modulators BAPTA (C-D) or TFP (E-F) under the different stress conditions indicated. For clarity, average values ± SD of the [Ca2+]c amplitudes and post-stimulatory [Ca2+]c resting levels are shown; however full Ca2+-signatures using the pretreatment with these modulators are plotted in S9 Fig Statistical analysis was performed using a 1-way ANOVA. * p < 0.05, ** p < 0.01, *** p < 0.005, **** p < 0.001.

Mentions: The maximum [Ca2+]c amplitude following application of the different stressors, and the post-stimulatory [Ca2+]c resting levels at 10 min after stressor application, were analysed in untreated, BAPTA (5 mM) and TFP (50 μM) pre-treated samples (Fig 4 and S9 Fig). A. fumigatus showed very different [Ca2+]c responses to each stressor with regard to [Ca2+]c amplitude and post-stimulatory [Ca2+]c resting levels (Fig 4 and S9 Fig). Pre-treatment with BAPTA or TFP radically altered [Ca2+]c responses in both stressor- and drug-dependent ways. To standardise comparisons between treatments, variations in maximal [Ca2+]c values were expressed as fold changes relative to challenge in the absence of drug (Fig 4C–4F). BAPTA pre-treatment significantly reduced the maximal [Ca2+]c amplitude after exposure to hypo-osmotic shock (5% growth medium), hyper-osmotic shock (500 mM mannitol), oxidative stress (20 mM H2O2) and 50% human serum (Fig 4C and S9 Fig). Thus the [Ca2+]c responses to these environmental challenges are likely to involve influx of extracellular Ca2+. The post-stimulatory [Ca2+]c resting level rarely changed significantly, except in the case of hypo-osmotic shock where a > 1 fold reduction, relative to pre-stimulatory resting level, was observed (Fig 4D). The effect of BAPTA on exposure to high extracellular Ca2+ was not performed because the use of a Ca2+-chelator is incompatible with this type of treatment.


Different Stress-Induced Calcium Signatures Are Reported by Aequorin-Mediated Calcium Measurements in Living Cells of Aspergillus fumigatus.

Muñoz A, Bertuzzi M, Bettgenhaeuser J, Iakobachvili N, Bignell EM, Read ND - PLoS ONE (2015)

Pretreatment with the Ca2+-chelator BAPTA or the calmodulin inhibitor TFP differentially impacts upon Ca2+-signalling and homeostasis during A. fumigatus responses to stressors.After growth for 20.5 h at 25°C, A. fumigatus AEQCEA10 cultures were pre-treated for 30 min with either 5 mM BAPTA or 50 μM TFP prior to challenge with stressors. [Ca2+]c amplitudes (measured simultaneously to stress application) and post-stimulatory [Ca2+]c resting levels (10 min after stress) are represented as [Ca2+]c values (A-B) and as fold change (Log2 ratios of treated/untreated) with the modulators BAPTA (C-D) or TFP (E-F) under the different stress conditions indicated. For clarity, average values ± SD of the [Ca2+]c amplitudes and post-stimulatory [Ca2+]c resting levels are shown; however full Ca2+-signatures using the pretreatment with these modulators are plotted in S9 Fig Statistical analysis was performed using a 1-way ANOVA. * p < 0.05, ** p < 0.01, *** p < 0.005, **** p < 0.001.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4581630&req=5

pone.0138008.g004: Pretreatment with the Ca2+-chelator BAPTA or the calmodulin inhibitor TFP differentially impacts upon Ca2+-signalling and homeostasis during A. fumigatus responses to stressors.After growth for 20.5 h at 25°C, A. fumigatus AEQCEA10 cultures were pre-treated for 30 min with either 5 mM BAPTA or 50 μM TFP prior to challenge with stressors. [Ca2+]c amplitudes (measured simultaneously to stress application) and post-stimulatory [Ca2+]c resting levels (10 min after stress) are represented as [Ca2+]c values (A-B) and as fold change (Log2 ratios of treated/untreated) with the modulators BAPTA (C-D) or TFP (E-F) under the different stress conditions indicated. For clarity, average values ± SD of the [Ca2+]c amplitudes and post-stimulatory [Ca2+]c resting levels are shown; however full Ca2+-signatures using the pretreatment with these modulators are plotted in S9 Fig Statistical analysis was performed using a 1-way ANOVA. * p < 0.05, ** p < 0.01, *** p < 0.005, **** p < 0.001.
Mentions: The maximum [Ca2+]c amplitude following application of the different stressors, and the post-stimulatory [Ca2+]c resting levels at 10 min after stressor application, were analysed in untreated, BAPTA (5 mM) and TFP (50 μM) pre-treated samples (Fig 4 and S9 Fig). A. fumigatus showed very different [Ca2+]c responses to each stressor with regard to [Ca2+]c amplitude and post-stimulatory [Ca2+]c resting levels (Fig 4 and S9 Fig). Pre-treatment with BAPTA or TFP radically altered [Ca2+]c responses in both stressor- and drug-dependent ways. To standardise comparisons between treatments, variations in maximal [Ca2+]c values were expressed as fold changes relative to challenge in the absence of drug (Fig 4C–4F). BAPTA pre-treatment significantly reduced the maximal [Ca2+]c amplitude after exposure to hypo-osmotic shock (5% growth medium), hyper-osmotic shock (500 mM mannitol), oxidative stress (20 mM H2O2) and 50% human serum (Fig 4C and S9 Fig). Thus the [Ca2+]c responses to these environmental challenges are likely to involve influx of extracellular Ca2+. The post-stimulatory [Ca2+]c resting level rarely changed significantly, except in the case of hypo-osmotic shock where a > 1 fold reduction, relative to pre-stimulatory resting level, was observed (Fig 4D). The effect of BAPTA on exposure to high extracellular Ca2+ was not performed because the use of a Ca2+-chelator is incompatible with this type of treatment.

Bottom Line: The 'signatures' of the transient [Ca2+]c responses to extracellular stimuli were found to be dose- and age-dependent.Moreover, Ca2+-signatures associated with each physico-chemical treatment were found to be unique, suggesting the involvement of heterogeneous combinations of Ca2+-signalling components in each stress response.The Ca2+-chelator BAPTA potently inhibited the initial responses to most stressors in accordance with a critical role for extracellular Ca2+ in initiating the stress responses.

View Article: PubMed Central - PubMed

Affiliation: Manchester Fungal Infection Group, Institute of Inflammation and Repair, University of Manchester, Manchester, United Kingdom; Fungal Cell Biology Group, Institute of Cell Biology, University of Edinburgh, Edinburgh, United Kingdom.

ABSTRACT
Aspergillus fumigatus is an inhaled fungal pathogen of human lungs, the developmental growth of which is reliant upon Ca2+-mediated signalling. Ca2+ signalling has regulatory significance in all eukaryotic cells but how A. fumigatus uses intracellular Ca2+ signals to respond to stresses imposed by the mammalian lung is poorly understood. In this work, A. fumigatus strains derived from the clinical isolate CEA10, and a non-homologous recombination mutant ΔakuBKU80, were engineered to express the bioluminescent Ca2+-reporter aequorin. An aequorin-mediated method for routine Ca2+ measurements during the early stages of colony initiation was successfully developed and dynamic changes in cytosolic free calcium ([Ca2+]c) in response to extracellular stimuli were measured. The response to extracellular challenges (hypo- and hyper-osmotic shock, mechanical perturbation, high extracellular Ca2+, oxidative stress or exposure to human serum) that the fungus might be exposed to during infection, were analysed in living conidial germlings. The 'signatures' of the transient [Ca2+]c responses to extracellular stimuli were found to be dose- and age-dependent. Moreover, Ca2+-signatures associated with each physico-chemical treatment were found to be unique, suggesting the involvement of heterogeneous combinations of Ca2+-signalling components in each stress response. Concordant with the involvement of Ca2+-calmodulin complexes in these Ca2+-mediated responses, the calmodulin inhibitor trifluoperazine (TFP) induced changes in the Ca2+-signatures to all the challenges. The Ca2+-chelator BAPTA potently inhibited the initial responses to most stressors in accordance with a critical role for extracellular Ca2+ in initiating the stress responses.

No MeSH data available.


Related in: MedlinePlus