Objectives: This study investigates how macrophages alter copper availability to control intracellular pathogens and the mechanisms by which Histoplasma capsulatum combats copper restriction.

Methods: A forward genetic screen of H. capsulatum identified loci required for proliferation within macrophages. Quantitative RT-PCR (qRT-PCR) was used to measure the transcriptional response of Histoplasma yeasts to high or low copper. To estimate intraphagosomal copper concentrations in macrophages, a gfp transcriptional reporter was used to measure the activity of the copper-responsive CTR3 promoter. Histoplasma yeasts lacking Ctr3 were tested for virulence using a murine model of pulmonary histoplasmosis. 

Results: The loss of the ability of Histoplasma yeast to replicate in macrophages was linked to disruption of the CTR3 locus, which encodes a fungal copper transporter. Loss of Ctr3 function impairs yeast growth in limiting copper consistent with Ctr3 functioning as a copper-specific importer. Transcription of the CTR3 gene is induced both by differentiation of H. capsulatum into pathogenic yeasts and by low available copper. Infection of macrophages with a strain having a transcriptional fusion to the CTR3 promoter  demonstrated that phagosomes of non-activated macrophages have moderate copper levels that are not antimicrobial to H. capsulatum. However, IFN-γ activation of phagocytes causes phagosomal copper restriction both in culture and in vivo. Accordingly, in a respiratory model of histoplasmosis, Ctr3-deficient yeasts are fully virulent during innate immunity but are attenuated after the onset of adaptive immunity.

Conclusion: Activation of macrophages during cell-mediated immunity, switches the phagosome from a copper-replete to a copper-depleted environment, forcing H. capsulatum reliance on Ctr3 for copper acquisition.

6 / Macrophage activation by IFN-? triggers restriction of phagosomal copper from intracellular pathogens