title: 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.
abstract: 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.
authors.affiliation: Departments of Pediatrics, Internal Medicine, and Medical Microbiology and Immunology, and the Comprehensive Cancer Center, University of Wisconsin Medical School, Madison, Wisconsin 53792, USA.
authors.forename: T
authors.initials: T
authors.lastname: Brandhorst
authors.forename: B
authors.initials: B
authors.lastname: Klein
mesh_terms: D000818:Animals; D001665:Binding Sites; D001692:Biological Transport; D001758:Blastomyces / Q000166:cytology / Q000378:metabolism* / Q000472:pathogenicity*; D015815:Cell Adhesion Molecules / Q000378:metabolism*; D002473:Cell Wall / Q000378:metabolism*; D002686:Chitin / Q000378:metabolism; D002687:Chitin Synthase / Q000235:genetics; D005656:Fungal Proteins; D006023:Glycoproteins / Q000378:metabolism*; D008264:Macrophages / Q000382:microbiology; D051379:Mice; D010587:Phagocytosis; D011485:Protein Binding; D020449:Repetitive Sequences, Amino Acid; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D015003:Yeasts / Q000166:cytology / Q000378:metabolism / Q000472:pathogenicity
chemical_list: D015815:Cell Adhesion Molecules; D005656:Fungal Proteins; D006023:Glycoproteins; D002686:Chitin; D002687:Chitin Synthase