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Aspergillus fumigatus  is an inhaled fungal pathogen of human lungs, the developmental growth of which is reliant upon Ca 2+ -mediated signalling. Ca 2+  signalling has regulatory significance in all eukaryotic cells but how  A .  fumigatus  uses intracellular Ca 2+  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  ΔakuB   KU80 , were engineered to express the bioluminescent Ca 2+ -reporter aequorin. An aequorin-mediated method for routine Ca 2+  measurements during the early stages of colony initiation was successfully developed and dynamic changes in cytosolic free calcium ([Ca 2+ ] c ) in response to extracellular stimuli were measured. The response to extracellular challenges (hypo- and hyper-osmotic shock, mechanical perturbation, high extracellular Ca 2+ , 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 [Ca 2+ ] c  responses to extracellular stimuli were found to be dose- and age-dependent. Moreover, Ca 2+ -signatures associated with each physico-chemical treatment were found to be unique, suggesting the involvement of heterogeneous combinations of Ca 2+ -signalling components in each stress response. Concordant with the involvement of Ca 2+ -calmodulin complexes in these Ca 2+ -mediated responses, the calmodulin inhibitor trifluoperazine (TFP) induced changes in the Ca 2+ -signatures to all the challenges. The Ca 2+ -chelator BAPTA potently inhibited the initial responses to most stressors in accordance with a critical role for extracellular Ca 2+  in initiating the stress responses.

Different Stress-Induced Calcium Signatures Are Reported by Aequorin-Mediated Calcium Measurements in Living Cells of Aspergillus fumigatus