Fibronectin-binding molecules of Scedosporium apiospermum: focus on adhesive events

Abstract

Scedosporium apiospermum is a widespread, emerging, and multidrug-resistant filamentous fungus that can cause localized and disseminated infections. The initial step in the infection process involves the adhesion of the fungus to host cells and/or extracellular matrix components. However, the mechanisms of adhesion involving surface molecules in S. apiospermum are not well understood. Previous studies have suggested that the binding of fungal receptors to fibronectin enhances its ability to attach to and infect host cells. The present study investigated the effects of fibronectin on adhesion events of S. apiospermum. The results revealed that conidial cells were able to bind to both immobilized and soluble human fibronectin in a typically dose-dependent manner. Moreover, fibronectin binding was virtually abolished in trypsin-treated conidia, suggesting the proteinaceous nature of the binding site. Western blotting assay, using fibronectin and anti-fibronectin antibody, evidenced 7 polypeptides with molecular masses ranging from 55 to 17 kDa in both conidial and mycelial extracts. Fibronectin-binding molecules were localized by immunofluorescence and immunocytochemistry microscopies at the cell wall and in intracellular compartments of S. apiospermum cells. Furthermore, a possible function for the fibronectin-like molecules of S. apiospermum in the interaction with host lung cells was assessed. Conidia pre-treated with soluble fibronectin showed a significant reduction in adhesion to either epithelial or fibroblast lung cells in a classically dose-dependent manner. Similarly, the pre-treatment of the lung cells with anti-fibronectin antibodies considerably diminished the adhesion. Collectively, the results demonstrated the presence of fibronectin-binding molecules in S. apiospermum cells and their role in adhesive events.

Keywords: Adhesion; Fibronectin; Infection; Lung cells; Scedosporium; Virulence.

Fig. 1

Binding of S. apiospermum conidia to soluble and immobilized human fibronectin. A Analysis of conidia binding to immobilized fibronectin by ELISA method. A solution of conidia (1 × 10⁶ cells) was added to fibronectin immobilized in 96-well polystyrene plates for 1 h at 37 °C. The reaction was developed using 3,3′,5,5′-tetramethylbenzidine and the color intensity was determined at 490 nm. Negative control wells were incubated only with BSA (400 µg/mL). B Analysis of conidia binding to soluble fibronectin by flow cytometry. Paraformaldehyde-fixed conidia (1 × 10⁶ cells) were sequentially incubated with soluble fibronectin at different concentrations, followed with anti-fibronectin antibody and FITC-labeled anti-IgG. Conidia treated only with the secondary antibody were used as control (autofluorescence). Trypsin treated conidial cells were also used before adding fibronectin in order to check the proteinaceous nature of the potential ligand. The results were expressed as mean of fluorescence intensity (MFI). The symbols indicate the experimental systems considered statistically significant from the control (P < 0.05; Student’s t-test)

Fig. 2

Localization of fibronectin-binding sites in S. apiospermum cells. A Detection of fibronectin-binding proteins in S. apiospermum conidial and mycelial whole extracts was carried out using a Western blotting assay. A control was included by incubating the membrane only with the secondary antibody, omitting the previous incubation with fibronectin. The numbers on the right refer to the molecular masses of standard proteins, expressed in kilodalton (kDa). B Fluorescence microscopy showing binding of soluble fibronectin to conidia (a phase-contrast microscopy; b fluorescence microscopy) and mycelia (c phase-contrast microscopy; d fluorescence microscopy) of S. apiospermum. Bars: 4 µm. C Immunocytochemical localization of fibronectin-binding sites in S. apiospermum conidia. Labeling is evident in intracellular compartments (arrowhead) and fungal cell wall (arrow). Bars: 4 µm (left image), 0.5 µm (center image), and 1 µm (right image)

Fig. 3

Participation of the fibronectin-binding molecules of S. apiospermum in the interaction with target host cells (epithelial and fibroblast lung cells). Conidia were incubated in the presence or absence of soluble human fibronectin at different concentrations (100–400 μg/ml) or with soluble BSA (400 μg/ml) at room temperature for 1 h. Alternatively, lung epithelial and fibroblast cells (A549 and MRC-5 lineages, respectively) were incubated with anti-fibronectin antibody at 1:100, 1:200, and 1:400 dilutions or with irrelevant IgG (1:400) for 1 h at 37 °C in an atmosphere with 5% CO₂. Posteriorly, fungi and lung cells were placed to interact in a proportion of 10:1, respectively, for 2 h at 37 °C at 5% CO₂. After the interaction period, the systems were washed and stained with Giemsa for subsequent determination of association indexes. The symbols indicate the experimental systems considered statistically significant from the control (P < 0.05; Student’s t-test)