Cell Wall Composition Heterogeneity between Single Cells in <named-content content-type="genus-species">Aspergillus fumigatus</named-content> Leads to Heterogeneous Behavior during Antifungal Treatment and Phagocytosis

ABSTRACT Aspergillus fumigatus can cause a variety of lung diseases in immunocompromised patients, including life-threatening invasive aspergillosis. There are only three main classes of antifungal drugs currently used to treat aspergillosis, and antifungal resistance is increasing. Experimental results in fungal biology research are usually obtained as average measurements across whole populations while ignoring what is happening at the single cell level. In this study, we show that conidia with the same genetic background in the same cell population at a similar developmental stage show heterogeneity in their cell wall labeling at the single cell level. We present a rigorous statistical method, newly applied to quantify the level of cell heterogeneity, which allows for direct comparison of the heterogeneity observed between treatments. We show the extent of cell wall labeling heterogeneity in dormant conidia and how the level of heterogeneity changes during germination. The degree of heterogeneity is influenced by deletions of cell wall synthesizing genes and environmental conditions, including medium composition, method of inoculation, age of conidia, and the presence of antifungals. This heterogeneity results in subpopulations of germinating conidia with heterogeneous fitness to the antifungal caspofungin, which targets cell wall synthesis and heterogeneous sensitivity of dormant conidia to phagocytosis by macrophages. IMPORTANCE The fungus Aspergillus fumigatus can cause invasive lung diseases in immunocompromised patients resulting in high mortality. Treatment using antifungal compounds is often unsuccessful. Average population measurements hide what is happening at the individual cell level. We set out to test what impact individual differences between the cell walls of fungal conidia have on their behavior. We show that a population of cells having the same genetic background gives rise to subpopulations of cells that exhibit distinct behavior (phenotypic heterogeneity). This cell heterogeneity is dependent on the strain type, gene deletions, cell age, and environmental conditions. By looking at the individual cell level, we discovered subpopulations of cells that show differential fitness during antifungal treatment and uptake by immune cells