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.

Fig 1. 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 AEQ^CEA10 strain. (B) Influence of the total amount of aequorin produced by fungal cells on the calculated pre-stimulatory resting [Ca²⁺]_c level.

Fig 2. Ca²⁺-signatures in response to mechanical perturbation and hypo-osmotic shock are growth dependent.

(A-C) The aequorin expressing strain AEQ^CEA10 was subjected to each stressor at various time-points of growth (18 to 24 h) at 25°C. Cultures were also microscopically analysed in order to compare the stage of conidial germination and germ tube growth with the [Ca²⁺]_c response. For clarity, average values for six technical replicates are shown without error bars; however the data is plotted with SD error bars in S6 Fig for comparison. The arrows indicate the point at which each stress was applied via the injectors of the plate reader. Bar: 10 μm.

Fig 3. Dose and stress-dependent Ca²⁺-signatures in response to (A) mannitol (hyper-osmotic shock), (B) high extracellular Ca²⁺, (C) exposure to H₂O₂ (oxidative stress), and (D) exposure to human serum.

After growth for 21 h at 25°C, A. fumigatus AEQ^CEA10 strain was challenged with different stressors applied at points indicated by arrows at the final concentrations shown in the Figure. For clarity, average values are shown; however technical replicates are plotted in S8 Fig.

Fig 4. Pretreatment with the Ca²⁺-chelator BAPTA or the calmodulin inhibitor TFP differentially impacts upon Ca²⁺-signalling and homeostasis during A. fumigatus responses to stressors.

After growth for 20.5 h at 25°C, A. fumigatus AEQ^CEA10 cultures were pre-treated for 30 min with either 5 mM BAPTA or 50 μM TFP prior to challenge with stressors. [Ca²⁺]_c amplitudes (measured simultaneously to stress application) and post-stimulatory [Ca²⁺]_c resting levels (10 min after stress) are represented as [Ca²⁺]_c values (A-B) and as fold change (Log₂ ratios of treated/untreated) with the modulators BAPTA (C-D) or TFP (E-F) under the different stress conditions indicated. For clarity, average values ± SD of the [Ca²⁺]_c amplitudes and post-stimulatory [Ca²⁺]_c resting levels are shown; however full Ca²⁺-signatures using the pretreatment with these modulators are plotted in S9 Fig Statistical analysis was performed using a 1-way ANOVA. * p < 0.05, ** p < 0.01, *** p < 0.005, **** p < 0.001.

Fig 5. 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 AEQ^CEA10 in AMM supplemented with 200 mM CaCl_2, 2.5 mM H₂O₂ and 50% human serum, 72 h at 25°C. (B) Optical density (OD₆₁₀) of A. fumigatus AEQ^CEA10, 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.