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

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.