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 ΔakuB KU80, 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.
Highlights
Aspergillus fumigatus causes multiple human and animal diseases, the manifestations of which depend upon a complex interplay between pathogen- and host-mediated activities [1,2,3]
To assess the utility of aequorin as a tool for measuring [Ca2+]c dynamics in A. fumigatus, we constructed a Ca2+-reporter strain in two different A. fumigatus genetic backgrounds, the wild-type clinical isolate CBS 144–89 (CEA10) and a non-homologous end re-joining mutant ΔakuBKU80, a null mutant in the AFUA_2G02620 locus, which exhibits significantly heightened homologous integration frequencies compared to non-mutated A. fumigatus isolates [34]
This vector contains a sequence of 1997 bp which is identical to that occurring immediately downstream of the AFUA_3G05360 stop codon, thereby directing targeted insertion of the plasmid into the A. fumigatus genome (S1 Fig)
Summary
Aspergillus fumigatus causes multiple human and animal diseases, the manifestations of which depend upon a complex interplay between pathogen- and host-mediated activities [1,2,3] Due to their small size, airborne spores (conidia) can penetrate deep into lung alveoli; in healthy individuals inhaled spores are likely to be efficiently silenced by coordinated innate immune mechanisms including the involvement of macrophages and neutrophils [4]. Antifungal defences are impaired in immunocompromised individuals (e.g. recipients of allogenic hematopoietic stem cell- or solid organ-transplants) in whom fungal colonies can become established following spore inhalation [5,6,7] This can give rise to the most lethal form of infection, invasive aspergillosis (IA), which annually causes around 200,000 deaths worldwide [8]. In comparison to colonised patients with similar underlying disease, CPA has been demonstrated to reach mortality rates of 42.8% over an observation period of 2 to 3 years [15]
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