TRANSFORMING DNA INTEGRATES AT MULTIPLE SITES IN THE DIMORPHIC FUNGALPATHOGEN BLASTOMYCES-DERMATITIDIS

Blastomyces dermatitidis is a primary fungal pathogen of man and other mammals, but like many other human fungal pathogens, relatively littl e is known about the factors that account for its virulence and pathog enicity. We developed a transformation system to facilitate molecular genetic studies of putative virulence factors from B. dermatitidis. Tr ansformation of the multinucleate yeasts was achieved by electroporati on of DNAs containing a dominant selectable marker, hygromycin B (HygB ) resistance. Southern analysis showed that transforming DNA invariabl y integrated ectopically into the chromosome. No evidence was found fo r extrachromosomal DNA. The HygB resistance could be expressed by eith er a 375-bp promoter fragment of the B. dermatitidis WI-1 gene encodin g adhesin or an Aspergillus gpdA promoter placed 5' of the E. coli hph gene. Primer extension analysis showed that for plasmids containing t he WI-1 promoter, transcription of the hph gene initiated within the 3 75-bp WI-1 promoter fragment. The combination of gene transfer and two promoters capable of independent transcription will allow us to resto re or augment gene expression in appropriate strains and test an influ ence on virulence. Molecular genetic manipulation of B. dermatitidis r epresents a major advance in our ability to investigate the pathogenes is of blastomycosis and other similar fungal diseases.