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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.

A serial enzymatic hydrolysis procedure for the partial lysis of Histoplasma capsulatum yeast-form cell walls was described, and its application for the differentiation of two serotypes was evaluated. Cell walls were serially digested with alpha(1 leads to 3)-glucanase and beta(1 leads to 3)-glucanse of Cladosporium resinae, then by Pronase, and then by chitinase. The walls of serotype 1, 2, 3 (61.5% digested) were not susceptible to alpha(1 leads to 3)-glucanse, and they contained 30.3% chitin, thus identifying the strain as chemotype 1 (chem 1). Serotype 1, 4 cells walls (51.6% digested) released 27.3% as glucose after treatment with alpha(1 leads to 3)-glucanse and contained 7.8% chitin, compatible with chemotype 2 (chem 2). In addition to quantitating the monomeric products of enzymolysis, I recovered soluble nondialyzable polysaccharide from the digests of both serotypes.

We have developed an improved scheme for the classification of environmental and clinical isolates of Histoplasma capsulatum that is based on analysis of mitochondrial DNA (mtDNA) and ribosomal DNA (rDNA). Strains were initially divided into mtDNA groups according to restriction digests of whole-cell DNA and Southern hybridization with cloned mtDNA probes. Strains within a mtDNA class could be further grouped by polymorphisms in rDNA. The majority of soil and clinical isolates from the United States had identical mtDNA patterns; however, rDNA polymorphisms were common in both types of isolates. The combination of mtDNA and rDNA typing described in this report will be useful in resolving questions concerning the epidemiology of H. capsulatum infections.