Identification and characterization of virulence factors in the fungal pathogen Histoplasma capsulatum

Histoplasma capsulatum, a fungal pathogen, colonizes macrophages during infection of mammalian hosts. In vitro studies showed that the fungus survives and replicates within the macrophage phagosome, ultimately lysing host cells. The fungal factors that promote these processes are largely unknown. Similarly, it is unknown whether a high intracellular fungal burden is sufficient to cause host cell lysis, or whether Histoplasma expresses factors that actively induce host cell death. To identify genes required for virulence in the macrophage, I designed a genetic screen to identify H. capsulatum mutants that failed to lyse macrophages. I tested 14,000 insertion mutants and identified twenty-six lysis-defective (LDF) mutants with a range of lysis defects and growth kinetics within macrophages. Several mutants appeared to be defective in genes that encode predicted secreted factors and metabolic proteins. Additionally, a number of the LDF genes had no orthologs in other fungal genomes, suggesting they play a role in Histoplasma-specific virulence mechanisms. One such gene, CBP1, had previously been identified in another lab as being required for lysis of macrophages and virulence in the mouse model of histoplasmosis. Surprisingly, the growth characteristics of the cbp1 mutant within macrophages had not previously been determined. I observed that Cbp1 is largely dispensable for intracellular growth within macrophages, but clearly required for macrophage lysis. These data provide the first evidence that a high intracellular fungal burden is not sufficient to trigger macrophage lysis.Since Cbp1 is a secreted factor, I reasoned that it might interact directly with host factors and modulate the host response. We used MEEBO microarrays to compare the transcriptional profile of macrophages infected with wild-type H. capsulatum and the cbp1 mutant. Cbp1 was required for the induction of a set of macrophage genes that was normally induced during infection with live H. capsulatum but not during infection with other intracellular pathogens. Since Cbp1 is also required for host-cell lysis, these data suggest that induction of the Cbp1-dependent transcriptional signature in macrophages is a molecular marker that correlates with host-cell death. Future work will address the molecular mechanism of Cbp1-dependent macrophage lysis