Regulated expression of BAD1, a virulence factor of Blastomyces dermatitidis

Dimorphic fungal pathogens exist as one morphotype in the environment, but convert to a host-adapted form upon infection. This conversion may be accompanied by the upregulation of virulence factors whose expression is limited to the pathogenic phase of the fungus, though few of these factors have been identified. In Blastomyces dermatitidis, a pathogen of humans, we examined the expression pattern of the virulence factor BAD1. BAD1 was expressed on yeast but not mycelia of all tested North American strains. No BAD1 transcript was detected in RNA from mycelia, and B. dermatitidis transformants harboring BAD1 promoter-lacZ fusions expressed beta-galactosidase reporter activity exclusively in yeast-phase cells. These data indicate that BAD1 promoter activity is transcriptionally regulated. Analysis of the BAD1 upstream region showed similarity to the sequence upstream of YPS3, a yeast phase-specific gene of the related fungus Histoplasma capsulatum . Two regions termed boxes B and A had the highest identity. To determine if the mechanism of phase-regulation was shared between these fungi, the BAD1 locus and BAD1-lacZ fusions were transformed into H. capsulatum. Expression of both BAD1 and the beta-galactosidase reporter was limited to the yeast phase, suggesting the mechanism of regulating transcription of yeast phase-specific genes is conserved.; To define functional regions within the promoter, serial truncations of BAD1 upstream sequences fused to lacZ were transformed into B. dermatitidis, and analyzed for reporter activity in yeast and mycelial phases. The 63-nucleotide box A region was required for BAD1 promoter activity. No significant mycelial-phase beta-galactosidase activity was recovered in BAD1-lacZ transformants, suggesting BAD1 transcription is positively regulated in yeast, not repressed in mycelia. B. dermatitidis transformants of matched YPS3-lacZ vectors showed a similar profile of expression, requiring box A for yeast-phase transcription. A subset of YPS3-lacZ and BAD1-lacZ constructs were analyzed in yeast phase H. capsulatum transformants. Box A was essential for BAD1 promoter activity, however the YPS3 promoter required additional sequence 5' to box A for transcriptional activity. Several potential transcription factor binding sites were identified within box A that may be involved in regulating phase-specific expression of BAD1 in B. dermatitidis and possibly other yeast phase-specific factors.