Cell wall biogenesis of Blastomyces dermatitidis. Evidence for a novel mechanism of cell surface localization of a virulence-associated adhesin via extracellular release and reassociation with cell wall chitin.

Pathogenic yeast of Blastomyces dermatitidis express a surface protein adhesin, WI-1. Due to the crucial role of WI-1 in adherence and disease pathogenesis, we investigated how the protein localizes to the surface of B. dermatitidis. WI-1 released extracellularly by wild-type yeast coated the surfaces of co-cultured knockout yeast within 3 h of incubation, implying that secreted WI-1 provides a pathway for loading the protein onto the yeast cell wall. In radioligand binding assays, purified WI-1 bound saturably, specifically, and with high affinity (K(d) = 8.3 x 10(-9)) to the cell surface of knockout yeast devoid of WI-1. WI-1 added exogenously, in vitro, to knockout yeast was indistinguishable from native cell surface WI-1 by fluorescence staining and restored adhesivity to the knockout yeast in macrophage binding and phagocytosis assays. Analysis of interactions between WI-1 and elements of the yeast cell wall identified chitin as the anchor point for WI-1. This interaction was shown to hinge on the 24-amino acid tandem repeat sequence of WI-1. Efforts to extract surface WI-1 from the yeast demonstrated that it is fastened to the wall by non-covalent interactions and covalent links between cysteine residues. We conclude that the yeast cell surface adhesin WI-1 localizes to the cell wall, in part, through extracellular release followed by high affinity binding back onto exposed chitin fibrils. These findings point to a novel pathway of cell wall biogenesis in yeast and an unanticipated role for chitin in anchoring and displaying a surface adhesin and virulence determinant.