Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, MO 63110, USA.
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 the saprophytic mycelial phase or the parasitic yeast phase. Recently, we identified a secreted calcium-binding protein, CBP, that is produced in large amounts by yeast cells but is undetectable in mycelial cultures. In this study, the green fluorescent protein (GFP) was established as a reporter in H. capsulatum to study regulation of CBP1 expression in cultures and in single 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 transition from mycelia to yeast, we demonstrated that the CBP1 promoter is only fully active after complete morphological conversion to the yeast form, indicating for the first time that CBP1 is developmentally regulated rather than simply temperature regulated. Continuous activity of the CBP1 promoter during infection of macrophages supports the hypothesis that CBP secretion plays an important role for Histoplasma survival within the phagolysosome. Broth cultures of Histoplasma yeasts carrying a CBP-GFP protein fusion construct were able to secrete a full-length fluorescent fusion protein that remained localized within the phagolysosomes of infected macrophages. Additionally, a comparison of two Histoplasma strains carrying the CBP1 promoter fusion construct either epichromosomally or integrated into the chromosome revealed cell-to-cell variation in plasmid copy number due to uneven plasmid partitioning into daughter cells.
Cell Microbiol. 2(6): 537-47.
Histoplasma capsulatum was transofomed with four versions of the natural wtGFP gene, GFPmut 2, SaGFP, LGFP, and sGFP, in order to increase the expression of GFP (Green fluorescence protein) in H. capsilatum. LGFP and sGFP were both codon optimized based on the codon usage of highly expressed Homo sapien genes. GFPmut 2 and SaGFP were codon optimized based on the codon usage patterns of E. coli and S. cerevisiae, respectively. GFPmut 2 and saGFP did not produce any GFP protein and LGFP only produced a weak fluroescene, but sGFP was able to produce fully functional and highly fluorescent GFP protein. Two of the four recoded genes were functional, but only one was able to produce a high functional amount of GFP. Overall, the recoded gene can be considered successful since the protein level and functionality increased and GFP was able to perform its reporter duty.
There were in total, 4 recoded genes used for this experiment, GFPmut 2, SaGFP, LGFP, and sGFP. There were no quantitative tests for the GFP protein levels, only fluorescence was detected.