Limits...
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

Dose and stress-dependent Ca2+-signatures in response to (A) mannitol (hyper-osmotic shock), (B) high extracellular Ca2+, (C) exposure to H2O2 (oxidative stress), and (D) exposure to human serum.After growth for 21 h at 25°C, A. fumigatus AEQCEA10 strain was challenged with different stressors applied at points indicated by arrows at the final concentrations shown in the Figure. For clarity, average values are shown; however technical replicates are plotted in S8 Fig.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4581630&req=5

pone.0138008.g003: Dose and stress-dependent Ca2+-signatures in response to (A) mannitol (hyper-osmotic shock), (B) high extracellular Ca2+, (C) exposure to H2O2 (oxidative stress), and (D) exposure to human serum.After growth for 21 h at 25°C, A. fumigatus AEQCEA10 strain was challenged with different stressors applied at points indicated by arrows at the final concentrations shown in the Figure. For clarity, average values are shown; however technical replicates are plotted in S8 Fig.

Mentions: Each stressor prompted immediate and distinctly different [Ca2+]c transients and thus different Ca2+-signatures (Fig 3 and S8 Fig), the amplitudes of which were dose-dependent with respect to each stressing stimulus. Following an initial rapid response to hyper-osmotic shock and serum treatments, there was a recovery of [Ca2+]c to the pre-stimulatory [Ca2+]c resting levels detected before the application of the stress stimulus. However, in response to high Ca2+ (5–200 mM) or oxidative stress (5–20 mM) the [Ca2+]c did not fully recover to the pre-stimulatory resting levels within 10 min of the initial application of the stress stimulus (see below). The highest [Ca2+]c amplitude was prompted by treatment with 200 mM extracellular CaCl2 (Fig 3B and S8 Fig), which provoked an increase in [Ca2+]c to ~ 0.9 μM. The magnitude of responses from high to low was highest for the high extracellular Ca2+ treatment and smallest for mannitol challenge (Ca2+ > H2O2 > serum > mannitol). Similar results were also achieved using the AEQΔakuB strain (data not shown).


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)

Dose and stress-dependent Ca2+-signatures in response to (A) mannitol (hyper-osmotic shock), (B) high extracellular Ca2+, (C) exposure to H2O2 (oxidative stress), and (D) exposure to human serum.After growth for 21 h at 25°C, A. fumigatus AEQCEA10 strain was challenged with different stressors applied at points indicated by arrows at the final concentrations shown in the Figure. For clarity, average values are shown; however technical replicates are plotted in S8 Fig.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0138008.g003: Dose and stress-dependent Ca2+-signatures in response to (A) mannitol (hyper-osmotic shock), (B) high extracellular Ca2+, (C) exposure to H2O2 (oxidative stress), and (D) exposure to human serum.After growth for 21 h at 25°C, A. fumigatus AEQCEA10 strain was challenged with different stressors applied at points indicated by arrows at the final concentrations shown in the Figure. For clarity, average values are shown; however technical replicates are plotted in S8 Fig.
Mentions: Each stressor prompted immediate and distinctly different [Ca2+]c transients and thus different Ca2+-signatures (Fig 3 and S8 Fig), the amplitudes of which were dose-dependent with respect to each stressing stimulus. Following an initial rapid response to hyper-osmotic shock and serum treatments, there was a recovery of [Ca2+]c to the pre-stimulatory [Ca2+]c resting levels detected before the application of the stress stimulus. However, in response to high Ca2+ (5–200 mM) or oxidative stress (5–20 mM) the [Ca2+]c did not fully recover to the pre-stimulatory resting levels within 10 min of the initial application of the stress stimulus (see below). The highest [Ca2+]c amplitude was prompted by treatment with 200 mM extracellular CaCl2 (Fig 3B and S8 Fig), which provoked an increase in [Ca2+]c to ~ 0.9 μM. The magnitude of responses from high to low was highest for the high extracellular Ca2+ treatment and smallest for mannitol challenge (Ca2+ > H2O2 > serum > mannitol). Similar results were also achieved using the AEQΔakuB strain (data not shown).

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