Insights into the interaction of Scedosporium apiospermum, Scedosporium aurantiacum, Scedosporium minutisporum, and Lomentospora prolificans with lung epithelial cells

Abstract

Scedosporium spp. and Lomentospora prolificans are filamentous fungi that emerged as human pathogens; however, their mechanisms of virulence/pathogenesis are still largely unknown. In the present work, we have evaluated the interaction of S. apiospermum, S. minutisporum, S. aurantiacum, and L. prolificans with lung epithelial cells (A549 line). The results showed that conidia were able to interact with A549 cells, displaying association indexes of 73.20, 117.98, 188.01, and 241.63 regarding S. apiospermum, L. prolificans, S. minutisporum, and S. aurantiacum, respectively. Light microscopy images evidenced morphological changes in epithelial cells, including rounding and detachment, especially during the interaction with L. prolificans. Plasma membrane injuries were detected in A549 cells after 1 h of co-culturing with S. aurantiacum and S. minutisporum and after 4 h with S. apiospermum and L. prolificans, as judged by the passive incorporation of propidium iodide. After 24 h of fungi-epithelial cells interaction, only mycelia were observed covering the A549 monolayer. Interestingly, the mycelial trap induced severe damage in the A549 cells, culminating in epithelial cell death. Our results demonstrate some relevant events that occur during the contact between lung epithelial cells and Scedosporium/Lomentospora species, including conidial adhesion and hyphal growth with consequent irreversible injury on A549 cells, adding light to the infection process caused by these opportunistic and multidrug-resistant fungi.

Keywords: Adhesion; Cellular interaction; Injury; Lomentospora; Lung epithelial cells; Scedosporium.

Fig. 1

In vitro co-culturing of human lung epithelial cells (A549 line) with S. apiospermum (Sap), S. minutisporum (Sm), S. aurantiacum (Sau), and L. prolificans (Lp). Conidia were interacted with epithelial cells in a ratio of 10:1. After 4 h, the association index was calculated as described in the “Materials and methods” section. The data are expressed as the mean ± standard deviation of three independent experiment sets performed in triplicate. All the systems were significantly different from each other

Fig. 2

Light micrographs showing the interaction events of A549 cells and Scedosporium/Lomentospora species. A549 cells were cultured in the absence (a) or in the presence of conidia of S. apiospermum (b), S. minutisporum (c), S. aurantiacum (d), and L. prolificans (e) for 1, 4, and 24 h. After 4 h of interaction, at ×100 magnification, it is possible to clearly detect morphological changes in A549 cells, except on S. apiospermum. Detachment of mammalian cells was seen mainly after the interaction with L. prolificans. The black arrows show the germinated conidia. Note the presence of a mycelial network covering the A549 cells after 24 h. Bars, 50 μm

Fig. 3

Fluorescence micrograph images showing the co-culture of Scedosporium/Lomentospora conidia and A549 cells. NHS-rhodamine-stained conidia and A549 cells (10:1) were interacted for 4 h at 37 °C. Subsequently, the plasma membrane and nucleus of A549 cells were stained with CT-B and DAPI, respectively. A control system containing only lung epithelial cells was used for comparison (a). Note that conidia of S. apiospermum (b), S. minutisporum (c), S. aurantiacum (d), and L. prolificans (e) randomly adhered to A549 cells. Bars, 50 μm

Fig. 4

Scedosporium/Lomentospora conidia induce plasma membrane damage in lung epithelial cells. A549 cells were cultured in the absence (a) or in the presence of conidia of S. apiospermum (b), S. minutisporum (c), S. aurantiacum (d), and L. prolificans (e) for 1, 2, 3, and 4 h. After each time point, the interaction systems were incubated with propidium iodide (PI), to evidence the A549 plasma membrane injury, and then visualized using phase-contrast (DIC) and fluorescence microscopy. Bars, 50 μm

Fig. 5

Microscopic analyses showing the mycelial trap covering the monolayer formed by lung epithelial cells. A549 cells were cultured in the absence (a) or in the presence of conidia of S. apiospermum (b), S. minutisporum (c), S. aurantiacum (d), and L. prolificans (e) after 24 h. Subsequently, the interaction systems were incubated with Calcofluor white (CW), to localize the mycelial cells, and propidium iodide (PI), to evidence the A549 plasma membrane injury. Note that after 24 h of co-culturing of fungal conidia and A549 cells, only mycelia were observed using phase-contrast (DIC) and fluorescence microscopy. Bars, 50 μm