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 virulen t 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 sequ ence. A 519-bp fragment of the cDNA containing the translational stop codon was linker modified and inserted into the bacterial expression v ector, pATH 1. Escherichia coli extracts containing the pATH 1 vector alone expressed a major 34-kDa TrpE polypeptide following induction wi th indoleacrylic acid, while the pATH 1/yps-3 construct produced a pre dominant 54-kDa TrpE/yps-3 fusion protein. Polyclonal rabbit sera dire cted against G217B reacted exclusively with the 54-kDa fusion protein in Western blots (immnoblots); 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 genera ted. A 17.4-kDa protein was detected with MAb 7.1 in Western blots pre pared from cell wall fractions of G217B; cytoplasmic fractions were un reactive. No yps-3 antigen was detected in either fraction of the Down s Strain, which fails to express the yps-3 gene. MAb 7.1 also detected a 17.4-kDa antigen in ethanol-precipitated culture supernatants deriv ed from G217B. These findings localize the yps-3 gene product to the c ell wall and culture supernatants, where the protein may influence the phase transition or the maintenance of the yeast state.