title: Different Stress-Induced Calcium Signatures Are Reported by Aequorin-Mediated Calcium Measurements in Living Cells of Aspergillus fumigatus.
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.
authors.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.
authors.forename: Alberto
authors.initials: A
authors.lastname: Muñoz
authors.affiliation: Manchester Fungal Infection Group, Institute of Inflammation and Repair, University of Manchester, Manchester, United Kingdom; Centre for Molecular Bacteriology and Infection, Department of Medicine, Imperial College London, London, United Kingdom.
authors.forename: Margherita
authors.initials: M
authors.lastname: Bertuzzi
authors.affiliation: Fungal Cell Biology Group, Institute of Cell Biology, University of Edinburgh, Edinburgh, United Kingdom.
authors.forename: Jan
authors.initials: J
authors.lastname: Bettgenhaeuser
authors.affiliation: Centre for Molecular Bacteriology and Infection, Department of Medicine, Imperial College London, London, United Kingdom.
authors.forename: Nino
authors.initials: N
authors.lastname: Iakobachvili
authors.affiliation: Manchester Fungal Infection Group, Institute of Inflammation and Repair, University of Manchester, Manchester, United Kingdom; Centre for Molecular Bacteriology and Infection, Department of Medicine, Imperial College London, London, United Kingdom.
authors.forename: Elaine M
authors.initials: EM
authors.lastname: Bignell
authors.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.
authors.forename: Nick D
authors.initials: ND
authors.lastname: Read
mesh_terms: D000331:Aequorin / Q000235:genetics / Q000378:metabolism*; D001232:Aspergillus fumigatus / Q000502:physiology*; D018533:Biomass; D002118:Calcium / Q000378:metabolism*; D002614:Chelating Agents / Q000494:pharmacology; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D015870:Gene Expression; D042541:Intracellular Space / Q000378:metabolism; D009997:Osmotic Pressure; D018384:Oxidative Stress; D010641:Phenotype; D013314:Stress, Mechanical; D013312:Stress, Physiological
chemical_list: D002614:Chelating Agents; D000331:Aequorin; D004533:Egtazic Acid; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; D002118:Calcium