Monitoring phase-specific gene expression in Histoplasma capsulatum with telomeric GFP fusion plasmids

Dimorphism is an essential feature of Histoplasma capsulatum pathogenesis, and much attention has been focused on characteristics that are unique to t he saprophytic mycelial phase or the parasitic yeast phase. Recently, we id entified a secreted calcium-binding protein, CBP, that is produced in large amounts by yeast cells but is undetectable in mycelial cultures, in this s tudy, the green fluorescent protein (GFP) was established as a reporter in H, capsulatum to study regulation of CBP1 expression in cultures and in sin gle cells grown under different conditions and inside macrophages, One GFP version that was optimized for human codon usage yielded highly fluorescent Histoplasma yeast cells, By monitoring GFP fluorescence during the transit ion from mycelia to yeast, we demonstrated that the CBP1 promoter is only f ully active after complete morphological conversion to the yeast form, indi cating for the first time that CBP1 is developmentally regulated rather tha n simply temperature regulated. Continuous activity of the CBP1 promoter du ring infection of macrophages supports the hypothesis that CBP secretion pl ays an important role for Histoplasma survival within the phagolysosome. Br oth cultures of Histoplasma yeasts carrying a CBP-GFP protein fusion constr uct were able to secrete a full-length fluorescent fusion protein that rema ined localized within the phagolysosomes of infected macrophages. Additiona lly, a comparison of two Histoplasma strains carrying the CBP1 promoter fus ion construct either epichromosomally or integrated into the chromosome rev ealed cell-to-cell variation in plasmid copy number due to uneven plasmid p artitioning into daughter cells.