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

The H antigen of the dimorphic fungal pathogen Histoplasma capsulatum was f irst described over 40 years ago. It is a secreted glycoprotein that is imm unogenic during infection. Recent cloning of the H antigen gene (HAG1) indi cated sequence homology with genes for fungal beta-glucosidases. To underst and the biological role of this immunodominant antigen in H. capsulatum, en zymatic 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 reve aled P-glucosidase activity near the predicted mobility of the H antigen. Q uantitative 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 e xhibited 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 be tween secreted enzyme activity and H protein levels. We took advantage of t hese class differences to demonstrate multicopy plasmid H gene overexpressi on by transformation of an HAG1 plasmid into H. capsulatum. Both a class II strain (G217Bura5-23) and a class III strain (G184ASura5-11) transformed,w ith 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 pro tein overexpression in this pathogenic fungus, and the findings support the identification of the H antigen as a beta-glucosidase.