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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

Increased intracellular aequorin correlates with the extent of conidial germination and fungal biomass.(A) Percentages of germination and total cytosolic aequorin present (in arbitrary relative light units, RLUs), as measured by using the aequorin discharge protocol (see Materials and Methods), for the AEQCEA10 strain. (B) Influence of the total amount of aequorin produced by fungal cells on the calculated pre-stimulatory resting [Ca2+]c level.
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pone.0138008.g001: Increased intracellular aequorin correlates with the extent of conidial germination and fungal biomass.(A) Percentages of germination and total cytosolic aequorin present (in arbitrary relative light units, RLUs), as measured by using the aequorin discharge protocol (see Materials and Methods), for the AEQCEA10 strain. (B) Influence of the total amount of aequorin produced by fungal cells on the calculated pre-stimulatory resting [Ca2+]c level.

Mentions: AEQCEA10 spore germination and germ tube growth results in a proportional increase in fungal biomass which is reflected by an increase in the total aequorin present in the cells (Fig 1A). Time-course analysis of AEQCEA10 spore germination for a period of 24 h at 25°C in AMM showed that > 80% of the conidia had germinated after 21 h of incubation (Fig 1A). This correlated with the maximal amount of aequorin expression in germlings, as measured by the maximum amount of aequorin luminescence detected (Fig 1A).


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)

Increased intracellular aequorin correlates with the extent of conidial germination and fungal biomass.(A) Percentages of germination and total cytosolic aequorin present (in arbitrary relative light units, RLUs), as measured by using the aequorin discharge protocol (see Materials and Methods), for the AEQCEA10 strain. (B) Influence of the total amount of aequorin produced by fungal cells on the calculated pre-stimulatory resting [Ca2+]c level.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0138008.g001: Increased intracellular aequorin correlates with the extent of conidial germination and fungal biomass.(A) Percentages of germination and total cytosolic aequorin present (in arbitrary relative light units, RLUs), as measured by using the aequorin discharge protocol (see Materials and Methods), for the AEQCEA10 strain. (B) Influence of the total amount of aequorin produced by fungal cells on the calculated pre-stimulatory resting [Ca2+]c level.
Mentions: AEQCEA10 spore germination and germ tube growth results in a proportional increase in fungal biomass which is reflected by an increase in the total aequorin present in the cells (Fig 1A). Time-course analysis of AEQCEA10 spore germination for a period of 24 h at 25°C in AMM showed that > 80% of the conidia had germinated after 21 h of incubation (Fig 1A). This correlated with the maximal amount of aequorin expression in germlings, as measured by the maximum amount of aequorin luminescence detected (Fig 1A).

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