title: 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.
authors.affiliation: Department of Medical Microbiology and Immunology, University of Wisconsin Medical School, Madison, Wisconsin 53706, USA.
authors.forename: K L
authors.initials: KL
authors.lastname: Fisher
authors.forename: G S
authors.initials: GS
authors.lastname: Deepe
authors.forename: J P
authors.initials: JP
authors.lastname: Woods
mesh_terms: D000818:Animals; D015966:Gene Expression Regulation, Fungal; D005800:Genes, Fungal; D014644:Genetic Variation; D006658:Histoplasma / Q000145:classification / Q000235:genetics* / Q000378:metabolism; D006659:Histoplasmin / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D011817:Rabbits; D001617:beta-Glucosidase / Q000235:genetics*
chemical_list: D006659:Histoplasmin; D001617:beta-Glucosidase