Histoplasma capsulatum strain variation in both H antigen production and beta-glucosidase activity and overexpression of HAG1 from a telomeric linear plasmid

Abstract

The H antigen of the dimorphic fungal pathogen Histoplasma capsulatum was first described over 40 years ago. It is a secreted glycoprotein that is immunogenic during infection. Recent cloning of the H antigen gene (HAG1) indicated sequence homology with genes for fungal beta-glucosidases. To understand the biological role of this immunodominant antigen in H. capsulatum, enzymatic assays were performed to determine whether H. capsulatum contained a beta-glucosidase enzyme activity and whether this activity was encoded by the HAG1 gene. Substrate gels with H. capsulatum culture supernatants revealed beta-glucosidase activity near the predicted mobility of the H antigen. Quantitative microtiter plate assays revealed marked differences in secreted beta-glucosidase activities from three H. capsulatum restriction fragment length polymorphism (RFLP) classes, with RFLP class II strains displaying high levels of enzyme activity, in contrast to the low levels of activity exhibited by class I and III strains. Immunoblotting of culture supernatants with an H antigen-specific antiserum demonstrated differences in H protein expression levels between the H. capsulatum classes, with a correlation between secreted enzyme activity and H protein levels. We took advantage of these class differences to demonstrate multicopy plasmid H gene overexpression by transformation of an HAG1 plasmid into H. capsulatum. Both a class II strain (G217Bura5-23) and a class III strain (G184ASura5-11) transformed with the telomeric overexpression plasmid pMAD401 displayed increased levels of beta-glucosidase enzyme activity and H protein expression compared to the levels in control transformants containing only the single genomic copy of HAG1. This is the first demonstration of telomeric plasmid-mediated protein overexpression in this pathogenic fungus, and the findings support the identification of the H antigen as a beta-glucosidase.

FIG. 1

Diagram of the telomeric HAG1 gene overexpression plasmid pMAD401 used in this study. Tel, H. capsulatum telomeric sequence comprised of repeats of the hexamer GGGTTA. Other plasmid elements are described in the text.

FIG. 2

Detection of β-glucosidase activity in H. capsulatum culture supernatants and demonstration that H antigen is present within the band of enzyme activity. (A) Nonreducing, nondenaturing gel separation of G217B supernatant protein (10 μg of protein) and almond β-glucosidase (2 μg). Yellow areas of substrate hydrolysis (here converted to gray scale) are marked with arrows. (B) Immunoblot of excised bands of enzyme activity reelectrophoresed following reduction and heat denaturation. Bands were electrophoresed alongside 10 μg of G217B supernatant protein as a positive control for the H antigen. A single immunoreactive band with a mobility similar to that of the H antigen was present in H. capsulatum (Hc) β-glucosidase. The difference in migration may be an effect of the gel slice preparation technique and reelectrophoresis. Molecular size markers (in kilodaltons) are indicated on the left.

FIG. 3

Microtiter plate analysis of β-glucosidase enzyme activity and H antigen-specific immunoblot analysis across H. capsulatum (Hc) RFLP classes. (A) H. capsulatum β-glucosidase enzyme activity was measured against an almond β-glucosidase standard curve. The x axis illustrates amounts of supernatant protein from each H. capsulatum strain; the y axis illustrates β-glucosidase enzyme activity (values are averages from duplicate wells). Different amounts of supernatant protein were used for different strains. Similar results were obtained in three independent experiments. (B) Immunoblot analysis of H antigen expression across H. capsulatum RFLP classes with the indicated amounts of supernatant protein. Molecular size markers (in kilodaltons) are indicated on the left.

FIG. 4

Demonstration of HAG1 gene overexpression by increased β-glucosidase enzyme activity and H antigen expression following H. capsulatum transformation with PmeI-digested pMAD401. (A) Microtiter plate assay results for parental strains, four pMAD401 transformants, and two control pWU45 transformants for both G184ASura5-11 and G217Bura5-23 lineages. For G184ASura5-11 and its derivatives (left panel), 10 μg of supernatant protein was tested. For G217Bura5-23 and its derivatives (right panel), 1 μg of supernatant protein was tested. The average β-glucosidase activity in duplicate wells from a representative experiment is shown. Similar results were observed in two independent experiments. (B) Immunoblot analysis of the same H. capsulatum strains as those shown in panel A with the indicated amounts of supernatant (SN) protein. Molecular size markers (in kilodaltons) are indicated on the left.