The fungal pathogenHistoplasma capsulatumparasitizes host phagocytes. To avoid antimicrobial immune responses,Histoplasmayeasts must minimize their detection by host receptors while simultaneously interacting with the phagocyte. PathogenicHistoplasmayeast cells, but not avirulent mycelial cells, secrete the Eng1 protein, which is a member of the glycosylhydrolase 81 (GH81) family. We show thatHistoplasmaEng1 is a glucanase that hydrolyzes β-(1,3)-glycosyl linkages but is not required forHistoplasmagrowthin vitroor for cell separation. However,Histoplasmayeasts lacking Eng1 function have attenuated virulencein vivo, particularly during the cell-mediated immunity stage.Histoplasmayeasts deficient for Eng1 show increased exposure of cell wall β-glucans, which results in enhanced binding to the Dectin-1 β-glucan receptor. Consistent with this, Eng1-deficient yeasts trigger increased tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6) cytokine production from macrophages and dendritic cells. While not responsible for large-scale cell wall structure and function, the secreted Eng1 reduces levels of exposed β-glucans at the yeast cell wall, thereby diminishing potential recognition by Dectin-1 and proinflammatory cytokine production by phagocytes. In α-glucan-producingHistoplasmastrains, Eng1 acts in concert with α-glucan to minimize β-glucan exposure: α-glucan provides a masking function by covering the β-glucan-rich cell wall, while Eng1 removes any remaining exposed β-glucans. Thus,HistoplasmaEng1 has evolved a specialized pathogenesis function to remove exposed β-glucans, thereby enhancing the ability of yeasts to escape detection by host phagocytes.IMPORTANCEThe success ofHistoplasma capsulatumas an intracellular pathogen results, in part, from an ability to minimize its detection by receptors on phagocytic cells of the immune system. In this study, we showed thatHistoplasmapathogenic yeast cells, but not avirulent mycelia, secrete a β-glucanase, Eng1, which reduces recognition of fungal cell wall β-glucans. We demonstrated that the Eng1 β-glucanase promotesHistoplasmavirulence by reducing levels of surface-exposed β-glucans on yeast cells, thereby enablingHistoplasmayeasts to escape detection by the host β-glucan receptor, Dectin-1. As a consequence, phagocyte recognition ofHistoplasmayeasts is reduced, leading to less proinflammatory cytokine production by phagocytes and less control ofHistoplasmainfectionin vivo. Thus,Histoplasmayeasts express two mechanisms to avoid phagocyte detection: masking of cell wall β-glucans by α-glucan and enzymatic removal of exposed β-glucans by the Eng1 β-glucanase.

Copper toxicity and copper limitation can both be effective host defense mechanisms against pathogens. Tolerance of high copper by fungi makes toxicity as a defense mechanism largely ineffective against fungal pathogens. A forward genetic screen for Histoplasma capsulatum mutant yeasts unable to replicate within macrophages showed the Ctr3 copper transporter is required for intramacrophage proliferation. Ctr3 mediates copper uptake and is required for growth in low copper. Transcription of the CTR3 gene is induced by differentiation of H. capsulatum into pathogenic yeasts and by low available copper, but not decreased iron. Low expression of a CTR3 transcriptional reporter by intracellular yeasts implies that phagosomes of non-activated macrophages have moderate copper levels. This is further supported by the replication of Ctr3-deficient yeasts within the phagosome of non-activated macrophages. However, IFN-γ activation of phagocytes causes restriction of phagosomal copper as shown by upregulation of the CTR3 transcriptional reporter and by the failure of Ctr3-deficient yeasts, but not Ctr3 expressing yeasts, to proliferate within these macrophages. Accordingly, in a respiratory model of histoplasmosis, Ctr3-deficient yeasts are fully virulent during phases of the innate immune response but are attenuated after the onset of adaptive immunity. Thus, while technical limitations prevent direct measurement of phagosomal copper concentrations and copper-independent factors can influence gene expression, both the CTR3 promoter induction and the attenuation of Ctr3-deficient yeasts indicate activation of macrophages switches the phagosome from a copper-replete to a copper-depleted environment, forcing H. capsulatum reliance on Ctr3 for copper acquisition. Control of primary pathogens that infect phagocytes often requires adaptive immunity, but the mechanisms that convert host cells from permissive to antimicrobial states are only partially understood. The intracellular fungal pathogen Histoplasma capsulatum resides and proliferates within the macrophage phagosome. During innate immunity, macrophages which normally control fungi prove ineffective against H. capsulatum yeasts. At this stage, the phagosome of unactivated macrophages has ample copper that facilitates intracellular growth of Histoplasma but does not cause copper toxicity. However, the onset of adaptive immunity and the subsequent activation of macrophages decreases phagosomal copper and macrophages become less permissive to Histoplasma proliferation. IFN-γ acts as a key cytokine for switching the macrophage strategy by changing phagosomes from a copper-sufficient to a copper-depleted state in order to control intracellular pathogens. In such activated macrophages, H. capsulatum yeasts upregulate expression of the Ctr3 copper transporter to enable continued acquisition of essential copper.