Macrophage activation by IFN-γ triggers restriction of phagosomal copper from intracellular pathogens

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.Author summary: 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.