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J Biol Chem, Vol. 275, Issue 11, 7925-7934, March 17, 2000
From the Departments of Pediatrics, Internal Medicine, and Medical
Microbiology and Immunology, and the Comprehensive Cancer Center,
University of Wisconsin Medical School, Madison, Wisconsin 53792
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
(Kd = 8.3 × 10
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*
and
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.
*
This work was supported in part by grants from the United
States Public Health Service.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Recipient of a Postdoctoral Training Fellowship from the American
Lung Association.
§
Recipient of National Institutes of Health Research Career
Development Award K04 AI01308 and a Burrroughs Wellcome Fund Scholar in
Molecular Pathogenic Mycology. To whom correspondence should be
addressed: University of Wisconsin-Madison, 600 Highland Ave., Rm.
K4/434, Madison, WI 53792. Tel.: 608-263-9217; Fax: 608-263-0440; E-mail: bsklein@facstaff.wisc.edu.
Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
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