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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

Growth of A. fumigatus is significantly impacted by the stressors and tolerance to oxidative stress is Ca2+ dependent.(A) Colonial growth phenotypes of a serial dilution (105, 104, 103 and 102 spores) of A. fumigatus AEQCEA10 in AMM supplemented with 200 mM CaCl2, 2.5 mM H2O2 and 50% human serum, 72 h at 25°C. (B) Optical density (OD610) of A. fumigatus AEQCEA10, measured in the presence or absence of BAPTA, following challenge with stressors. Prior to the application of the challenge indicated, cultures were grown at 25°C for 21 h, or 20.5 h if pre-treatment with BAPTA was applied. Following challenge, growth was allowed to commence for a further 72 h before measurements were taken. Statistical significance was calculated using 2-way ANOVA to compare each challenge, in the presence or absence of BAPTA, to the respective unchallenged measurements. * p < 0.05, ** p < 0.01, *** p < 0.005, **** p < 0.001.
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pone.0138008.g005: Growth of A. fumigatus is significantly impacted by the stressors and tolerance to oxidative stress is Ca2+ dependent.(A) Colonial growth phenotypes of a serial dilution (105, 104, 103 and 102 spores) of A. fumigatus AEQCEA10 in AMM supplemented with 200 mM CaCl2, 2.5 mM H2O2 and 50% human serum, 72 h at 25°C. (B) Optical density (OD610) of A. fumigatus AEQCEA10, measured in the presence or absence of BAPTA, following challenge with stressors. Prior to the application of the challenge indicated, cultures were grown at 25°C for 21 h, or 20.5 h if pre-treatment with BAPTA was applied. Following challenge, growth was allowed to commence for a further 72 h before measurements were taken. Statistical significance was calculated using 2-way ANOVA to compare each challenge, in the presence or absence of BAPTA, to the respective unchallenged measurements. * p < 0.05, ** p < 0.01, *** p < 0.005, **** p < 0.001.

Mentions: In order to assess the effects of stressors upon A. fumigatus growth, and the role of extracellular Ca2+ in adapting to such challenges, a phenotypic growth assay was implemented (Fig 5). To assess colonial phenotypes the different stressors were added to solid AMM medium, serial dilutions of spores (105, 104, 103 and 102 spores) in 5 μl of water were inoculated and colonial size and morphology evaluated after 72 h of growth at 25°C (Fig 5A). In a separate analysis aimed at understanding stresses applied in the same multiwell plate liquid AMM environment utilised for aequorin-mediated measurement of Ca2+-signalling (Fig 5B), the different stressors were applied to untreated and BAPTA pre-treated 21 h-old fungal cultures, and their growth monitored over the next 72 h. All stressors prompted significant growth perturbations when applied on solid (Fig 5A) or in liquid (Fig 5B) media. Growth was greatly enhanced by addition of 50% human serum, consistent with one or more components of this complex substrate providing nutrients for the growing pathogen (Fig 5B). This observation was also supported by analyses of radial growth which indicated more robust growth in response to serum (Fig 5A). In contrast, fungal growth was highly sensitive to 2.5 mM H2O2 and concentrations higher than 2.5 mM were found to be toxic to the cells in the 21–72 h period between stimulation and growth measurement (Fig 5). Highlighting the importance of extracellular Ca2+ for A. fumigatus growth (Loss and Bertuzzi et al., submitted), BAPTA treatment decreased hyphal growth rate in liquid culture in the absence of a challenge. Most importantly, consistent with a role for extracellular Ca2+ in mediating the response of A. fumigatus to these stresses, BAPTA treatment significantly perturbed hyphal growth rate in liquid culture when hyphae were challenged with H2O2 and 50% human serum (Fig 5B). In the presence of BAPTA, oxidative stress and human serum caused approximately a 10 fold decrease and a 2 fold increase respectively, relative to unchallenged A. fumigatus fungal growth (Fig 5B). Similar experiments were also performed in the presence of the calmodulin inhibitor TFP. However, no significant inhibition/modulation of the fungal growth by TFP was observed during the 21–72 h period (data not shown). This observation of the differential growth inhibitory effect of BAPTA and TFP is consistent with our observations that showed a greater impairment of the [Ca2+]c responses when BAPTA was added (Fig 4C and 4D) in comparison to the TFP pretreatment (Fig 4E and 4F).


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)

Growth of A. fumigatus is significantly impacted by the stressors and tolerance to oxidative stress is Ca2+ dependent.(A) Colonial growth phenotypes of a serial dilution (105, 104, 103 and 102 spores) of A. fumigatus AEQCEA10 in AMM supplemented with 200 mM CaCl2, 2.5 mM H2O2 and 50% human serum, 72 h at 25°C. (B) Optical density (OD610) of A. fumigatus AEQCEA10, measured in the presence or absence of BAPTA, following challenge with stressors. Prior to the application of the challenge indicated, cultures were grown at 25°C for 21 h, or 20.5 h if pre-treatment with BAPTA was applied. Following challenge, growth was allowed to commence for a further 72 h before measurements were taken. Statistical significance was calculated using 2-way ANOVA to compare each challenge, in the presence or absence of BAPTA, to the respective unchallenged measurements. * p < 0.05, ** p < 0.01, *** p < 0.005, **** p < 0.001.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0138008.g005: Growth of A. fumigatus is significantly impacted by the stressors and tolerance to oxidative stress is Ca2+ dependent.(A) Colonial growth phenotypes of a serial dilution (105, 104, 103 and 102 spores) of A. fumigatus AEQCEA10 in AMM supplemented with 200 mM CaCl2, 2.5 mM H2O2 and 50% human serum, 72 h at 25°C. (B) Optical density (OD610) of A. fumigatus AEQCEA10, measured in the presence or absence of BAPTA, following challenge with stressors. Prior to the application of the challenge indicated, cultures were grown at 25°C for 21 h, or 20.5 h if pre-treatment with BAPTA was applied. Following challenge, growth was allowed to commence for a further 72 h before measurements were taken. Statistical significance was calculated using 2-way ANOVA to compare each challenge, in the presence or absence of BAPTA, to the respective unchallenged measurements. * p < 0.05, ** p < 0.01, *** p < 0.005, **** p < 0.001.
Mentions: In order to assess the effects of stressors upon A. fumigatus growth, and the role of extracellular Ca2+ in adapting to such challenges, a phenotypic growth assay was implemented (Fig 5). To assess colonial phenotypes the different stressors were added to solid AMM medium, serial dilutions of spores (105, 104, 103 and 102 spores) in 5 μl of water were inoculated and colonial size and morphology evaluated after 72 h of growth at 25°C (Fig 5A). In a separate analysis aimed at understanding stresses applied in the same multiwell plate liquid AMM environment utilised for aequorin-mediated measurement of Ca2+-signalling (Fig 5B), the different stressors were applied to untreated and BAPTA pre-treated 21 h-old fungal cultures, and their growth monitored over the next 72 h. All stressors prompted significant growth perturbations when applied on solid (Fig 5A) or in liquid (Fig 5B) media. Growth was greatly enhanced by addition of 50% human serum, consistent with one or more components of this complex substrate providing nutrients for the growing pathogen (Fig 5B). This observation was also supported by analyses of radial growth which indicated more robust growth in response to serum (Fig 5A). In contrast, fungal growth was highly sensitive to 2.5 mM H2O2 and concentrations higher than 2.5 mM were found to be toxic to the cells in the 21–72 h period between stimulation and growth measurement (Fig 5). Highlighting the importance of extracellular Ca2+ for A. fumigatus growth (Loss and Bertuzzi et al., submitted), BAPTA treatment decreased hyphal growth rate in liquid culture in the absence of a challenge. Most importantly, consistent with a role for extracellular Ca2+ in mediating the response of A. fumigatus to these stresses, BAPTA treatment significantly perturbed hyphal growth rate in liquid culture when hyphae were challenged with H2O2 and 50% human serum (Fig 5B). In the presence of BAPTA, oxidative stress and human serum caused approximately a 10 fold decrease and a 2 fold increase respectively, relative to unchallenged A. fumigatus fungal growth (Fig 5B). Similar experiments were also performed in the presence of the calmodulin inhibitor TFP. However, no significant inhibition/modulation of the fungal growth by TFP was observed during the 21–72 h period (data not shown). This observation of the differential growth inhibitory effect of BAPTA and TFP is consistent with our observations that showed a greater impairment of the [Ca2+]c responses when BAPTA was added (Fig 4C and 4D) in comparison to the TFP pretreatment (Fig 4E and 4F).

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