GENOMIC CLONING, CHARACTERIZATION, AND FUNCTIONAL-ANALYSIS OF THE MAJOR SURFACE ADHESIN WI-1 ON BLASTOMYCES-DERMATITIDIS YEASTS

WI-1 is a 120-kDa surface protein adhesin on Blastomyces dermatitidis yeasts that binds CD18 and CD14 receptors on human macrophages. We iso lated and analyzed a clone of genomic WI-1 to characterize this key ad herence mechanism of the yeast. The 9.3-kilobase insert contains an op en reading frame of 3438 nucleotides and no introns. The amino acid se quence of native WI-1 matches the deduced sequence of genomic WI-1 at positions 757-769, 901-913, and 1119-1138, demonstrating the cloned ge ne is authentic WI-1. The complete coding sequence has 30 highly conse rved repeats of 24 amino acids arrayed in tandem in two noncontiguous regions of the protein. The repeat sequence is homologous to the Yersi niae adhesin invasin, the C terminus displays an epidermal growth fact or-like domain, and the N terminus has a short hydrophobic sequence th at may be a membrane spanning domain. The tandem repeats are predicted to be at the exposed surface of the protein, thereby explaining the a dhesive properties of WI-1. The WI-1 promoter contains a CAAT box (nuc leotide positions 2287-2290), TATA box (2380-2385), and CT motif (2399 -2508). Transcription is initiated within the CT motif at nucleotide 2 431. A 5.5-kilobase subclone containing the full coding sequence of WI -1 was expressed as a histidine-tagged fusion protein in Escherichia c oli. Recombinant WI-1 has the expected molecular mass of 120 kDa, is s trongly recognized in Western blots by rabbit anti-WI-1 antiserum, and binds human macrophage receptors in the same manner as native WI-1. T his work clarifies a key adherence mechanism of B. dermatitidis and wi ll permit further analysis of WI-1-mediated attachment to host cells, receptors, and extracellular matrix.