Discovery of a role for Hsp82 in Histoplasma virulence through a quantitative screen for macrophage lethality.

The application of forward genetics can reveal new factors required for the virulence of intracellular pathogens. To facilitate such virulence screens, we developed macrophage cell lines with which the number of intact host cells following infection with intracellular pathogens can be rapidly and easily ascertained through the expression of a constitutive lacZ transgene. Using known virulence mutants of Francisella novicida and Histoplasma capsulatum, we confirmed the applicability of these host cells for the quantitative assessment of bacterial and fungal virulence, respectively. To identify new genes required for Histoplasma virulence, we employed these transgenic macrophage cells to screen a collection of individual transfer DNA (T-DNA) insertion mutants. Among the mutants showing decreased virulence in macrophages, we identified an insertion in the locus encoding the Histoplasma Hsp82 homolog. The lesion caused by the T-DNA insertion localizes to the promoter region, resulting in significantly decreased HSP82 expression. Reduced HSP82 expression markedly attenuates the virulence of Histoplasma yeast in vivo. While the HSP82 hypomorph grows normally in vitro at 37°C and under acid and salinity stresses, its ability to recover from high-temperature stress is impaired. These results provide genetic proof of the role of stress chaperones in the virulence of a thermally dimorphic fungal pathogen.