Abstract
The precise characterization of the mechanisms modulating Aspergillus fumigatus survival within airway epithelial cells has been impaired by the lack of live-cell imaging technologies and user-friendly quantification approaches. Here we described the use of an automated image analysis pipeline to estimate the proportion of A. fumigatus spores taken up by airway epithelial cells, those contained within phagolysosomes or acidified phagosomes, along with the fungal factors contributing to these processes. Coupling the use of fluorescent A. fumigatus strains and fluorescent epithelial probes targeting lysosomes, acidified compartments and cell membrane, we found that both the efficacy of lysosome recruitment to phagosomes and phagosome acidification determines the capacity of airway epithelial cells to contain A. fumigatus growth. Overall, the capability of the airway epithelium to prevent A. fumigatus survival was higher in bronchial epithelial than alveolar epithelial cells. Certain A. fumigatus cell wall mutants influenced phagosome maturation in airway epithelial cells. Taken together, this live-cell 4D imaging approach allows observation and measurement of the very early processes of A. fumigatus interaction within live airway epithelial monolayers.
Highlights
Introduction published maps and institutional affilMore than 10 million people globally suffer from lung diseases caused by the environmental fungus Aspergillus fumigatus [1], and over 200,000 annually die from it [2].Inhalation of A. fumigatus spores is constant and unavoidable for all human beings [3].Early after exposure, A. fumigatus spores are eliminated from the airways by the innate lung defences [4,5,6]
To quantify lysosome trafficking to phagosomes containing A. fumigatus spores, the expression of a GFP construct fused to the lysosomal marker LAMP1 was monitored for 1 h in A549 lung epithelial cells
Since a small proportion of A. fumigatus spores can germinate and escape from the host cell [35], it is likely that survival of spores and germlings within phagolysosomes is determined by both, the number of free lysosomes present in the epithelial cell cytoplasm and their rate of fusion with the phagosome
Summary
More than 10 million people globally suffer from lung diseases caused by the environmental fungus Aspergillus fumigatus [1], and over 200,000 annually die from it [2]. A. fumigatus spores are eliminated from the airways by the innate lung defences [4,5,6]. The extended use of immunosuppressive treatments alongside the extremely large population suffering from chronic lung conditions and severe respiratory infections has placed A. fumigatus as the major respiratory mould pathogen in patients with debilitated lung defences [7,8,9,10,11]. Intraphagosomal killing of A. fumigatus spores by professional phagocytes such as macrophages is the most efficient process to prevent fungal persistence and the development of disease [12,13,14,15]. Epithelial cells vastly outnumber macrophages in the normal lung and an increasing body of evidence has shown that uptake of adherent iations
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