Ultrastructural viewpoints on the interaction events of Scedosporium apiospermum conidia with lung and macrophage cells

Abstract

Background: Scedosporium apiospermum is a ubiquitous, emerging and multidrug-resistant fungal pathogen with still rather unknown virulence mechanisms.

Objectives/methods: The cellular basis of the in vitro interaction between fungi and host cells/tissues is the determinant factor for the development of a successful in vivo infection. Herein, we evaluated the interaction of S. apiospermum conidia with lung epithelial (A549), lung fibroblast (MRC-5) and RAW 264.7 macrophages by light and scanning/transmission electron microscopy.

Findings: After 4 h of fungi-host cell contact, the percentage of infected mammalian cells and the number of fungi per infected cell was measured by light microscopy, and the following association indexes were calculated for A549, MRC-5 and macrophage cells: 73.2 ± 25.9, 69.7 ± 22.5 and 59.7 ± 11.1, respectively. Both conidia and germinated conidia were regularly observed interacting with the evaluated cells, with a higher prevalence of non-germinated conidia. Interestingly, nests of germinated conidia were evidenced at the surface of lung cells by scanning electron microscopy. Some germination projections and hyphae were seen penetrating/evading the mammalian cells. Furthermore, internalised conidia were seen within vacuoles as visualised by transmission electron microscopy.

Main conclusions: The present study contributes to a better understanding of S. apiospermum pathogenesis by demonstrating the first steps of the infection process of this opportunistic fungus.

Fig. 1:. interaction of Scedosporium apiospermum with A549, MRC-5 and RAW mammalian cells for 4 h at 37ºC. The association indices of S. apiospermum with A549 alveolar pulmonary cells, MRC-5 fibroblast pulmonary cells and RAW murine macrophages (A); the percentage of infected A549, MRC-5 and RAW cells (B); and the mean number of fungi per infected cell (C) are shown. The data are expressed as the mean ± standard deviation (SD) of three independent experimental sets.

Fig. 2:. Scedosporium apiospermum morphotypes observed during the interaction process with A549, MRC-5 and RAW mammalian cells after 4 h at 37ºC. (A) Percentages of distinct morphotypes, germinated and non-germinated conidia interacting with A549, MRC-5 and RAW cells. The data are expressed as the mean ± standard deviation (SD) of three independent experimental sets. The diamonds in (A) represent the significant differences between the germinated and non-germinated conidia in each group (p < 0.05). (B) In the bright field microscopy, open circles demonstrate non-germinated conidia, white arrows show host cells with several internalised conidia, and black arrows point to germinated conidia. The mammalian cells submitted to the interaction process did not show visible morphological changes, with the exception of RAW cells, which became more sprawling after contact with the fungus. The micrographs are representative images of three independent experimental sets.

Fig. 3:. scanning electron microscopy (SEM) of the interaction between Scedosporium apiospermum conidia and A549 cells. The images revealed many conidial cells adhered to animal cells and some germinated conidia (A-E). Thin white arrows indicate the associated conidia; the white arrowhead shows dispersed germinated conidia, while the open circles show “nests” of germinated conidia. The thick white arrow points to a hollow corresponding to the exact location where the conidium was anchored to the mammalian cell (B). A filamentous network produced by epithelial cells can be seen surrounding germinated conidia (E). The micrographs are representative images of three independent experimental sets.

Fig. 4:. scanning electron microscopy (SEM) of the interaction between Scedosporium apiospermum conidia and MRC-5 fibroblasts. Fungal cells were placed in contact with the animal cells for 4 h. Conidial cells adhered to animal cells can be observed (A, B, C), as well as many grouped germinated conidia on the surface of the fibroblasts (D). In E, F and G, it is possible to see hyphae penetrating into the animal cells forming holes and cavities (white arrows) corresponding to the invasion sites. The micrographs are representative images of three independent experimental sets.

Fig. 5:. scanning electron microscopy (SEM) of the interaction between Scedosporium apiospermum conidia and RAW macrophages. Conidia, germinated conidia and hyphae appear simultaneously on and inside the phagocytes. RAW macrophages can be seen touching the conidia at an initial stage (asterisks - A, E, F) or at a later stage, where the conidia appear almost completely phagocytosed (arrowhead - B). A phagocytosed conidium can be seen breaking the macrophage cytoplasmic membrane and reaching the extracellular medium via germination growth (circle - C). The white arrows denote macrophages engulfing the fungal hyphae through extensions of their cytoplasm (D - G). E, F and G show more than one macrophage trying to hold the same long hyphae. The magnification in H shows the possible breaking of a hyphae caused by phagocytosis by two macrophages (open arrow). The micrographs are representative images of three independent experimental sets.

Fig. 6:. transmission electron microscopy (TEM) of the interaction between Scedosporium apiospermum conidia and A549 epithelial cells (A-D), MRC-5 fibroblasts (E-H) and RAW (I-L) macrophages. Internalised conidia can be viewed in the absence (A, F) or in the presence of a vacuole (E, J, K). The vacuole membrane can be visualised (white arrow). Black arrows show fungi inside the vacuole. B, F and I show the exact moment that conidia invade the animal cell (white asterisks). In C and L, a conidial cell is seen inside a late endosome, already in the degradation process (white arrowhead). Note that in D, G and H the presence of adhered fungi to the animal cells. PM indicates the location of the plasma membrane of the animal cells, whereas CW shows the fungal cell wall. The micrographs are representative images of three independent experimental sets.