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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

Ca2+-signatures in response to mechanical perturbation and hypo-osmotic shock are growth dependent.(A-C) The aequorin expressing strain AEQCEA10 was subjected to each stressor at various time-points of growth (18 to 24 h) at 25°C. Cultures were also microscopically analysed in order to compare the stage of conidial germination and germ tube growth with the [Ca2+]c response. For clarity, average values for six technical replicates are shown without error bars; however the data is plotted with SD error bars in S6 Fig for comparison. The arrows indicate the point at which each stress was applied via the injectors of the plate reader. Bar: 10 μm.
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pone.0138008.g002: Ca2+-signatures in response to mechanical perturbation and hypo-osmotic shock are growth dependent.(A-C) The aequorin expressing strain AEQCEA10 was subjected to each stressor at various time-points of growth (18 to 24 h) at 25°C. Cultures were also microscopically analysed in order to compare the stage of conidial germination and germ tube growth with the [Ca2+]c response. For clarity, average values for six technical replicates are shown without error bars; however the data is plotted with SD error bars in S6 Fig for comparison. The arrows indicate the point at which each stress was applied via the injectors of the plate reader. Bar: 10 μm.

Mentions: Previous studies involving aequorin-based, multiwell plate luminometry measurements in A. awamori and N. crassa have demonstrated that transient [Ca2+]c increases, with reproducible Ca2+-signatures, are induced when these fungi are: (i) mechanically perturbed (by a rapid injection of iso-osmotic growth medium); (ii) exposed to hypo-osmotic shock (by injecting diluted 5% growth medium); or (iii) treated with a high (0.05–50 mM) concentration of external Ca2+ (by injecting growth medium containing these different concentrations of Ca2+ [23,31,41–43]. In this study, a plate luminometer incorporated into a multimode plate reader, and having built-in injectors, was utilised to subject A. fumigatus aequorin-expressing isolates to similar physiochemical perturbations. Each of these environmental challenges was found to produce a unique, and reproducible, Ca2+-signature in A. fumigatus (Fig 2 and S6 Fig).


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)

Ca2+-signatures in response to mechanical perturbation and hypo-osmotic shock are growth dependent.(A-C) The aequorin expressing strain AEQCEA10 was subjected to each stressor at various time-points of growth (18 to 24 h) at 25°C. Cultures were also microscopically analysed in order to compare the stage of conidial germination and germ tube growth with the [Ca2+]c response. For clarity, average values for six technical replicates are shown without error bars; however the data is plotted with SD error bars in S6 Fig for comparison. The arrows indicate the point at which each stress was applied via the injectors of the plate reader. Bar: 10 μm.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0138008.g002: Ca2+-signatures in response to mechanical perturbation and hypo-osmotic shock are growth dependent.(A-C) The aequorin expressing strain AEQCEA10 was subjected to each stressor at various time-points of growth (18 to 24 h) at 25°C. Cultures were also microscopically analysed in order to compare the stage of conidial germination and germ tube growth with the [Ca2+]c response. For clarity, average values for six technical replicates are shown without error bars; however the data is plotted with SD error bars in S6 Fig for comparison. The arrows indicate the point at which each stress was applied via the injectors of the plate reader. Bar: 10 μm.
Mentions: Previous studies involving aequorin-based, multiwell plate luminometry measurements in A. awamori and N. crassa have demonstrated that transient [Ca2+]c increases, with reproducible Ca2+-signatures, are induced when these fungi are: (i) mechanically perturbed (by a rapid injection of iso-osmotic growth medium); (ii) exposed to hypo-osmotic shock (by injecting diluted 5% growth medium); or (iii) treated with a high (0.05–50 mM) concentration of external Ca2+ (by injecting growth medium containing these different concentrations of Ca2+ [23,31,41–43]. In this study, a plate luminometer incorporated into a multimode plate reader, and having built-in injectors, was utilised to subject A. fumigatus aequorin-expressing isolates to similar physiochemical perturbations. Each of these environmental challenges was found to produce a unique, and reproducible, Ca2+-signature in A. fumigatus (Fig 2 and S6 Fig).

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