TY - JOUR
T1 - Localization of a yeast-phase-specific gene product to the cell wall in Histoplasma capsulatum
AU - Weaver, Carol H.
AU - Sheehan, Kathleen C.F.
AU - Keath, Elizabeth J.
PY - 1996
Y1 - 1996
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=0030054510&partnerID=8YFLogxK
U2 - 10.1128/iai.64.8.3048-3054.1996
DO - 10.1128/iai.64.8.3048-3054.1996
M3 - Article
C2 - 8757832
AN - SCOPUS:0030054510
SN - 0019-9567
VL - 64
SP - 3048
EP - 3054
JO - Infection and immunity
JF - Infection and immunity
IS - 8
ER -