Discovery of a role for Hsp82 in Histoplasma virulence through a quantitative screen for macrophage lethality
Jessica A Edwards1, Olga Zemska, Chad A Rappleye
1Department of Microbiology, The Center for Microbial Interface Biology, Ohio State University, Columbus, OH 43210, USA.
Abstract:
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.
Insights
Researchers developed transgenic macrophage cell lines for rapid virulence screening of intracellular pathogens. They identified a key role for Histoplasma Hsp82 in fungal virulence and host cell survival.
Area of Science:
- Microbiology
- Genetics
- Immunology
Background:
- Forward genetics is crucial for identifying virulence factors in intracellular pathogens.
- Quantitative assessment of pathogen virulence requires robust host-cell-based screening systems.
Purpose of the Study:
- To develop and validate transgenic macrophage cell lines for high-throughput virulence screening.
- To identify novel host factors contributing to the virulence of intracellular pathogens, specifically Histoplasma.
Main Methods:
- Development of constitutive lacZ-expressing macrophage cell lines for quantifying host cell integrity post-infection.
- Validation using known virulence mutants of Francisella novicida (bacterial) and Histoplasma capsulatum (fungal).
- Screening of Histoplasma T-DNA insertion mutants using the developed transgenic macrophage system.
Main Results:
- The transgenic macrophage system accurately quantified virulence for both bacterial and fungal pathogens.
- A Histoplasma mutant with a T-DNA insertion in the HSP82 promoter region exhibited significantly reduced virulence.
- Reduced HSP82 expression impaired fungal virulence in vivo and thermotolerance, despite normal in vitro growth.
Conclusions:
- Transgenic macrophage cell lines provide an effective platform for quantitative virulence screening.
- Hsp82 is a critical determinant of Histoplasma virulence, particularly in response to thermal stress.
- Stress chaperones play a vital role in the pathogenicity of thermally dimorphic fungi.
