Abstract
WI-1 is a 120-kDa surface protein adhesin on Blastomyces dermatitidis yeasts that binds CD18 and CD14 receptors on human macrophages. We isolated and analyzed a clone of genomic WI-1 to characterize this key adherence mechanism of the yeast. The 9.3-kilobase insert contains an open reading frame of 3438 nucleotides and no introns. The amino acid sequence of native WI-1 matches the deduced sequence of genomic WI-1 at positions 757-769, 901-913, and 1119-1138, demonstrating the cloned gene is authentic WI-1. The complete coding sequence has 30 highly conserved repeats of 24 amino acids arrayed in tandem in two noncontiguous regions of the protein. The repeat sequence is homologous to the Yersiniae adhesin invasin, the C terminus displays an epidermal growth factor-like domain, and the N terminus has a short hydrophobic sequence that may be a membrane-spanning domain. The tandem repeats are predicted to be at the exposed surface of the protein, thereby explaining the adhesive properties of WI-1. The WI-1 promoter contains a CAAT box (nucleotide positions 2287-2290), TATA box (2380-2385), and CT motif (2399-2508). Transcription is initiated within the CT motif at nucleotide 2431. A 5.5-kilobase subclone containing the full coding sequence of WI-1 was expressed as a histidine-tagged fusion protein in Escherichia coli. Recombinant WI-1 has the expected molecular mass of 120 kDa, is strongly recognized in Western blots by rabbit anti-WI-1 antiserum, and binds human macrophage receptors in the same manner as native WI-1. This work clarifies a key adherence mechanism of B. dermatitidis and will permit further analysis of WI-1-mediated attachment to host cells, receptors, and extracellular matrix.
Highlights
Important fungi pose a serious health hazard, especially for the growing numbers of immunocompromised patients worldwide [1]
The work below was accomplished with the published 1-kb cDNA, designated BK1, and an additional larger WI-1 cDNA isolated from strain 26199, designated p1.1.2 cDNA p1.1, which overlaps with 477 nucleotides at the 5Ј end of cDNA BK1, extends further than BK1 toward the 5Ј end of WI-1 coding sequence
Because cDNA p1.1 contained WI-1 coding sequence missing from cDNA BK1, we used cDNA p1.1 to further analyze genomic WI-1 by Southern analysis. cDNA p1.1 was used to screen a genomic library made from DNA of strain 26199 to eliminate problems associated with sequence variation
Summary
Important fungi pose a serious health hazard, especially for the growing numbers of immunocompromised patients worldwide [1]. Binding studies demonstrated WI-1 is an adhesin, which mediates attachment of yeasts to macrophages; the 25-amino acid repeat interacts with both CR3 and CD14 receptors [15]. In view of the importance of adherence in understanding and intervening in the pathogenesis of invasive fungal diseases, and because of the key role of WI-1 in binding yeasts to host tissues, we sought to characterize WI-1 more carefully at the molecular level. We show the striking finding that the full-length gene contains 30 copies of the invasin-like repeat within the coding sequence. This information sheds new light on a key adherence mechanism in this pathogenic fungus
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