Isolation and partial characterization of an adhesin from Fonsecaea pedrosoi.

We showed previously that mannose and N-acetylglucosamine (GlcNAc) residues are involved in the process of adhesion of Fonsecaea pedrosoi, the causative agent of chromoblastomycosis, to epithelial cells. It was then suggested that lectin-like molecules would be involved in the interaction. In the present study, we used fluorescein isothiocyanate-labeled neoglycoproteins (bovine serum albumin [BSA]-mannose and BSA-GlcNAc) to analyze the presence of sugar-binding proteins on the surface of conidia of F. pedrosoi grown at 28 and 37 degrees C. Binding of the neoglycoproteins was measured using flow cytometry. Fungal conidia expressed high levels of binding sites for BSA-mannose and BSA-GlcNAc when grown at 37 degrees C rather than 28 degrees C. Binding was inhibited by previous incubation of the conidia in the presence of chloroquine and trypsin. Chloroquine treatment also inhibited the interaction of fungal conidia with Chinese hamster ovary cells. Extracts from the conidia, obtained using a mechanical cell homogenizer, were purified by affinity chromatography using mannose-agarose or GlcNAc-agarose column. Polyacrylamide gel electrophoresis of the purified material from both columns showed a single protein band of 50 kDa, suggesting that the same lectin-like protein recognizes both carbohydrates.

The influence of surface carbohydrates on the interaction of Fonsecaea pedrosoi with Chinese hamster ovary glycosylation mutant cells.

In order to better understand the role played by surface glycoconjugates during cell adhesion and endocytosis by the dematiaceous fungi Fonsecaea pedrosoi, we analyzed the interaction between this microorganism and five mutants of Chinese Hamster Ovary (CHO) cells, which differ from each other in the exposition of carbohydrate residues on the cell surface. Five clones (Gat-2 parental, and the clones: Lec1, Lec2, Lec8 and ldlLec1) were tested and the adhesion and endocytic indexes were determined after 2 hours of interaction. The Lec1 and ldlLec1 clones, which present exposed mannose residues, showed the greater adhesion index (AI). On the other hand, the Lec8 clone, which exposes N-acetylglucosamine on the cell surface, presented the greater endocytic index. The role played by surface-exposed carbohydrate residues was further analyzed by addition of mannose or N-acetylglucosamine to the interaction medium and by previous incubation of the cells in the presence of the lectins Concanavalin A (ConA) and wheat germ agglutinin (WGA). The results obtained suggest that mannose residues are involved in the first step of adhesion of F. pedrosoi to the cell surface, while N-acetylglucosamine residues are involved on its ingestion process.