Proteome analysis of programmed cell death in Aspergillus fumigatus conidia as basis for a "dead or alive" reporter strain

Conidia produced by the human-pathogenic fungus Aspergillus fumigatus are the main cause of invasive aspergillosis in immunocompromised patients. In the lung, alveolar macrophages phagocytose the inhaled conidia and process them intracellularly, thereby clearing them from the airways. However, the fungus interferes with this mechanism to avoid being killed and antifungal compounds are needed to treat the infection and support the immune system. However, many currently available drugs exhibit adverse side effects, and the development of new therapeutic strategies is urgently needed. Now, it is challenging to distinguish between resting and dead conidia in situ, as they do not show easily detectable metabolic or morphological differences. The generation of a reporter strain that produces a cell death‑associated protein fused with the green fluorescent protein would facilitate the differentiation between dead and living conidia by fluorescence microscopy. To identify a suitable reporter protein, we investigated the induction of programmed cell death in A. fumigatus conidia. An in vitro cell death assay was established by treating conidia with various cell death-inducing compounds, testing different concentrations, time points and cultivation media. The results demonstrated that H2O2 in a nutrient-rich medium kills resting conidia better than the antifungal drugs amphotericin B or voriconazole. On the basis of these data, the optimal conditions for proteome analysis were determined. By comparing the proteome of resting, swollen and dying conidia, we identified eleven proteins which are differentially abundant during regulated cell death. The further analysis of these proteins will provide mechanistic insights into programmed cell death in A. fumigatus conidia. Moreover, these proteins were used to generate GFP reporter strains and their potential use as an indicator of conidial cell death is to be tested in co-incubation studies of conidia with RAW 264.7 macrophages.