Recognition of fibronectin by Penicillium marneffei conidia via a sialic acid-dependent process and its relationship to the interaction between conidia and laminin

Abstract

Adhesion of Penicillium marneffei conidia to the extracellular matrix protein laminin via a sialic acid-dependent process has previously been demonstrated. This study describes the interaction of P. marneffei conidia with fibronectin and examines the relationship of this process to the recognition of laminin via conidia. Immunofluorescence microscopy demonstrated that fibronectin bound to the surface of conidia and to phialides, but not to hyphae, in a pattern similar to that reported for laminin. Conidia were able to bind to fibronectin immobilized on microtiter plates in a concentration-dependent manner. However, binding to fibronectin (at any given concentration of protein and conidia) was less than that to laminin under equivalent conditions. Soluble fibronectin and antifibronectin antibody inhibited adherence of conidia to fibronectin in the plate adherence assay; soluble laminin also caused pronounced inhibition. Various monosaccharides and several peptides had no effect on adherence to fibronectin. However, N-acetylneuraminic acid abolished adherence to fibronectin, indicating that the interaction was mediated through a sialic acid-dependent process; the latter parallels observations of laminin binding by conidia. Fibronectin binding (and binding of laminin) was considerably reduced by prolonged preincubation of conidia with chymotrypsin, suggesting the protein nature of the binding site. Conidia from older cultures were more adherent to both immobilized fibronectin and laminin than conidia from younger cultures. Ligand affinity binding demonstrated the presence of a 20-kDa protein with the ability to bind both fibronectin and laminin. There would therefore appear to be a common receptor for the binding of fibronectin and laminin on the surface of P. marneffei, and the interaction described here maybe important in mediating attachment of the fungus to host tissue.

FIG. 1

Immunofluorescence identification of the binding of fibronectin. (a and b) Phase-contrast and immunofluorescence microscopy of conidia and hyphae incubated with fibronectin, antifibronectin antibody, and FITC-labelled conjugate. Negatively staining hyphae are arrowed. (c and d) Phase-contrast and immunofluorescence microscopy of phialides incubated as described above. Bars represent 10 μm.

FIG. 2

Attachment of P. marneffei conidia to immobilized fibronectin (solid bars) and laminin (hatched bars) at a range of protein concentrations (0.1 to 500 μg/ml). Conidia concentration was constant at 10⁵ per well. Results, expressed as the number of adherent conidia for 10 fields, are the means of triplicate counts performed three times (with standard deviations included) and are shown in the same manner in Fig. 3 to 6.

FIG. 3

Inhibition of attachment of P. marneffei conidia to immobilized fibronectin by soluble ligand and specific antibody. Wells were coated with a 100-μg/ml fibronectin solution, and conidia were allowed to adhere after preincubation in PBS (A), BSA (1 mg/ml) (B), soluble fibronectin (500 μg/ml [C] and 1 mg/ml [D]), soluble laminin (500 μg) (E), and (1 mg/ml) (F). Conidia were also coincubated in the presence of antifibronectin antibody (1:50) (G), antilaminin antibody (1:50) (H), RGD peptide (I), and YIGSR peptide (J). The values in the presence of soluble fibronectin, laminin, and antifibronectin antibody were significantly different from the value with PBS (P < 0.05).

FIG. 4

Inhibition of attachment of P. marneffei conidia to immobilized fibronectin by sialic acid. Wells were coated with a 100-μg/ml fibronectin solution, and conidia were allowed to adhere after preincubation in PBS (A), galactose (B), mannose (C), glucose (D), asialomucin (E), mucin (F), and sialic acid (G). The value in the presence of sialic acid was significantly different from the value with PBS (P < 0.01).

FIG. 5

Effect on adherence of P. marneffei conidia to immobilized fibronectin after preincubation with chymotrypsin (at 1,000 μg/ml) with time. Open bars, controls not incubated with chymotrypsin; solid bars, samples incubated with chymotrypsin and bound to fibronectin.

FIG. 6

Effect of ageing of P. marneffei conidia on adherence to fibronectin (solid bars) and to laminin (hatched bars).

FIG. 7

Identification by ligand affinity blotting of proteins from P. marneffei conidia which bind fibronectin and laminin. Lanes: A, whole conidial homogenate stained with silver stain; B, immunoblotting of lane A with fibronectin, antifibronectin antibody, and peroxidase conjugate; C, immunoblotting of lane A with laminin, antilaminin antibody, and peroxidase conjugate. Relative molecular masses are shown on the left in kilodaltons.