The influence of a copper efflux pump in Histoplasma capsulatum virulence

During the infectious process, pathogenic microorganisms must obtain nutrients from the host in order to survive and proliferate. These nutritional sources include the metallic nutrient copper. Despite its essentiality, copper in large amounts is toxic. Host defense mechanisms use high copper poisoning as a fungicidal strategy to control infection. Transcriptional analyses showed that yeast cultured in the presence of copper or inside macrophages (24 h) had elevated expression of CRP1, a copper efflux pump, suggesting that Histoplasma capsulatum could be exposed to a high copper environment in macrophages during the innate immune stage of infection. Accordingly, macrophages cultured in high copper are more efficient in controlling H. capsulatum growth. Also, silencing of ATP7a, a copper pump that promotes the copper influx in phagosomes, increases fungal survival in macrophages. The rich copper environment faced by the fungus is not dependent on IFN‐γ, since fungal CRP1 expression is induced in untreated macrophages. Appropriately, CRP1 knockdown fungal strains are more susceptible to macrophage control than wild‐type yeasts. Additionally, CRP1 silencing decreases fungal burden in mice during the phase of innate immune response (4‐day postinfection) and CRP1 is required for full virulence in a macrophage cell lines (J774 A.1 and RAW 264.7), as well as primary cells (BMDM). Thus, induction of fungal copper detoxifying genes during innate immunity and the attenuated virulence of CRP1‐knockdown yeasts suggest that H. capsulatum is exposed to a copper‐rich environment at early infection, but circumvents this condition to establish infection. Histoplasma capsulatum elevates Crp1 expression in the presence of copper in vitro or in macrophage infection. Copper chelation hampered macrophage ability to control the fungus, while fungal burden decreases in copper‐treated immune cells. ATP7a silencing (phagosome copper pump) also caused increased fungal burden. H. capsulatum CRP1‐knockdown strains attenuated virulence. These data show that copper increase is a strategy for phagocytes to control H. capsulatum, and Crp1 is responsible for H. capsulatum virulence.