Despite the emerging clinical importance of invasive fungal diseases in normal and immune-compromised hosts, the molecular basis of host-fungal pathogen interactions remains poorly understood. The dimorphic fungus Blastomyces dermatitidis displays a 120-kD surface protein (designated WI- 1) that mediates adhesion of the yeast to mammalian cells. Five- to tenfold more WI-1 is displayed on the surface of genetically related attenuated and avirulent mutant yeasts than on the parental wild-type yeast, but shedding of WI-1 is impaired in the mutants. We hypothesize that WI-1 normally promotes infection by adhering the fungus to nonphagocytic cells or cell matrix, but that WI-1 must be regulated at sites of inflammation to avoid immune recognition. Following adherence, the fungus avoids phagocyte recognition by masking or shedding surface WI- 1. Defects in these strategies reduce virulence by permitting enhanced phagocyte binding of WI-1, and consequently, phagocytosis and killing of yeasts. This application has two goals. They are to study the role of WI-1 in mediating interaction of B. dermatitidis with human macrophages and their receptors, and to identify the WI-1 domains that mediate the interactions.
The specific aims are: 1) To define the influence of enhanced WI-1 surface expression and impaired shedding on the interaction of B. dermatitidis with macrophages. The genetically related strains of B. dermatitidis with defined alterations of WI-1 will be compared for recognition, phagocytosis, and killing in vitro by human macrophages. The roles of WI- 1, macrophage CD18 and CD14 receptors and serum opsonins such as complement in dictating recognition of the different strains will be investigated. 2) To localize receptor binding domains of WI-1. Deletion derivatives of WI-1 will be produced by unidirectional digestion of the full-length gene. Deletion products will be coated on latex beads to study binding to macrophages and their CD18 and CD14 receptors. WI-1 domains that mediate binding will be defined more precisely with synthetic peptides. In further studies, WI-1 Mab that block attachment of cells to native WI-1 will be used to l) define epitopes recognized by probing the deletion products in western blots, and 2) demonstrate that these epitopes are exposed and mediate binding in their native configuration on yeasts. This work will establish a paradigm for understanding the molecular basis of fungal attachment, a vital mechanism of pathogenesis that is poorly understood in B. dermatitidis and related fungi. Furthermore, dissecting how natural perturbations in this process alter fungal pathogenicity may lead directly to new avenues for preventing and treating infections with medically important fungi.
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