Immunologic recognition of a 25-amino acid repeat arrayed in tandem on a major antigen of Blastomyces dermatitidis.
A 120-kD glycoprotein antigen abundantly expressed on Blastomyces dermatitidis yeasts is a target of cellular and humoral immune responses in human infection. To investigate the antigen and immune response more carefully at the molecular level, we screened an expression library from B. dermatitidis to identify clones that encode this antigen, designated WI-1. A 942-bp cDNA was isolated by immunologic screening with polyclonal, rabbit anti-WI-1 antiserum. Northern hybridization analysis showed that the cDNA hybridized to yeast message approximately equal to 3.9 kb. DNA and deduced protein sequence analysis of the clone demonstrated a 25-amino acid repeat arrayed in tandem, present in 4.5 copies near the 5' end, and rich in predicted antigenic epitopes. Further analysis showed strong homology in these tandem repeats with invasin, an adhesin of Yersiniae. Cloned cDNA was used to express a 30-kD fusion protein strongly recognized in western blots by rabbit anti-WI-1 antiserum, and by sera from all 35 blastomycosis patients studied. The fusion protein product of subcloned cDNA encoding only the tandem repeat also was strongly recognized in western blots by sera from the 35 blastomycosis patients, but not by sera from 10 histoplasmosis and 5 coccidioidomycosis patients. An antigen-inhibition radioimmunoassay showed that the tandem repeat alone completely eliminated rabbit and human anti-WI-1 antibody binding to radiolabeled native WI-1. From these results, we conclude that the 25-amino acid repeat of WI-1 displays an immunodominant B cell epitope, and that the carboxyl-terminus of the molecule exhibits an architecture that may promote adhesion of Blastomyces yeasts to host cells or extracellular matrix proteins and ultimately provide a clearer picture of the molecular pathogenesis of blastomycosis.
Klein
B S
BS
Department of Pediatrics, University of Wisconsin Medical School, Madison.
Hogan
L H
LH
Jones
J M
JM
D000595:Amino Acid Sequence; D000908:Antibodies, Fungal / Q000276:immunology; D000946:Antigens, Fungal / Q000737:chemistry / Q000276:immunology*; D001483:Base Sequence; D001758:Blastomyces / Q000737:chemistry / Q000276:immunology*; D001759:Blastomycosis / Q000276:immunology*; D003001:Cloning, Molecular; D016384:Consensus Sequence; D004247:DNA / Q000235:genetics; D005656:Fungal Proteins; D006023:Glycoproteins / Q000235:genetics* / Q000276:immunology; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D011994:Recombinant Proteins / Q000276:immunology; D017386:Sequence Homology, Amino Acid
D000908:Antibodies, Fungal; D000946:Antigens, Fungal; D005656:Fungal Proteins; D006023:Glycoproteins; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D004247:DNA