Localization of a yeast-phase-specific gene product to the cell wall in Histoplasma capsulatum.
A yeast-phase-specific gene, yps-3, has been identified in the virulent Histoplasma capsulatum strain, G217B. Although DNA sequencing of the genomic yps-3 gene from G217B failed to detect homologies with known proteins, the 5' end of a yps-3 cDNA contained a consensus signal sequence. A 519-bp fragment of the cDNA containing the translational stop codon was linker modified and inserted into the bacterial expression vector, pATH 1. Escherichia coli extracts containing the pATH 1 vector alone expressed a major 34-kDa TrpE polypeptide following induction with indoleacrylic acid, while the pATH 1/yps-3 construct produced a predominant 54-kDa TrpE/yps-3 fusion protein. Polyclonal rabbit sera directed against G217B reacted exclusively with the 54-kDa fusion protein in Western blots (immunoblots); serum samples from three patients with acute pulmonary or disseminated histoplasmosis were also positive. To localize the yps-3 protein within G217B, a monoclonal antibody (MAb 7.1) which recognized the yps-3 portion of the fusion protein was generated. A 17.4-kDa protein was detected with MAb 7.1 in Western blots prepared from cell wall fractions of G217B; cytoplasmic fractions were unreactive. No yps-3 antigen was detected in either fraction of the Downs strain, which fails to express the yps-3 gene. MAb 7.1 also detected a 17.4-kDa antigen in ethanol-precipitated culture supernatants derived from G217B. These findings localize the yps-3 gene product to the cell wall and culture supernatants, where the protein may influence the phase transition or the maintenance of the yeast state.
Weaver
C H
CH
Department of Biology, Saint Louis University, Missouri 63103, USA.
Sheehan
K C
KC
Keath
E J
EJ
D000818:Animals; D000908:Antibodies, Fungal; D000911:Antibodies, Monoclonal; D015153:Blotting, Western; D002458:Cell Fractionation; D002473:Cell Wall / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D005798:Genes, Bacterial; D006658:Histoplasma / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D006660:Histoplasmosis / Q000097:blood; D006801:Humans; D009024:Morphogenesis; D017433:Protein Structure, Secondary; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000276:immunology; D017421:Sequence Analysis; D015003:Yeasts
D000908:Antibodies, Fungal; D000911:Antibodies, Monoclonal; D018076:DNA, Complementary; D005656:Fungal Proteins; D011993:Recombinant Fusion Proteins; C087923:Yps-3 protein, Ajellomyces capsulata