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 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.
Hogan
L H
LH
Department of Pediatrics, University of Wisconsin Medical School, University of Wisconsin Hospital and Clinics, Madision 53792, USA.
Josvai
S
S
Klein
B S
BS
D000595:Amino Acid Sequence; D000818:Animals; D015703:Antigens, CD / Q000502:physiology*; D000946:Antigens, Fungal / Q000276:immunology*; D001483:Base Sequence; D001758:Blastomyces / Q000235:genetics / Q000276:immunology*; D018821:CD18 Antigens / Q000502:physiology*; D003001:Cloning, Molecular; D017124:Conserved Sequence; D004926:Escherichia coli; D005656:Fungal Proteins; D015870:Gene Expression; D005800:Genes, Fungal; D006023:Glycoproteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology*; D006801:Humans; D018950:Lipopolysaccharide Receptors / Q000502:physiology*; D008264:Macrophages / Q000276:immunology*; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D011401:Promoter Regions, Genetic; D011817:Rabbits; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000276:immunology / Q000302:isolation & purification; D012091:Repetitive Sequences, Nucleic Acid; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D016385:TATA Box
D015703:Antigens, CD; D000946:Antigens, Fungal; D018821:CD18 Antigens; D005656:Fungal Proteins; D006023:Glycoproteins; D018950:Lipopolysaccharide Receptors; D011994:Recombinant Proteins