PROJECT SUMMARY Control of primary fungal pathogens such as Histoplasma capsulatum requires the adaptive immune response to activate macrophages. Without macrophage activation, Histoplasma yeasts invade and thrive within these host cells. Following activation, macrophages become restrictive to fungal growth, however the mechanisms underlying this change are only partially known. Preliminary work shows that transition to this restrictive state correlates with a switch from phagosomes containing high concentrations of copper to a copper-limited state, which imposes nutritional limitations to fungal growth. This proposal will develop and optimize the necessary methodology to isolate Histoplasma-containing compartments from the macrophage to facilitate biochemical studies of this important macrophage organelle. This will enable determination of the trace element and protein composition of the Histoplasma-containing phagosome. Phagosomal copper dynamics and the cytokine signals that regulate phagosome copper levels will be confirmed in vivo using fluorescent Histoplasma yeasts as a live probe of phagosomal copper availability. Comparative protein analyses between phagosomes from Histoplasma-permissive macrophages and from Histoplasma-restrictive macrophages will be performed to identify candidate host factors that mediate the activation-dependent limitation of phagosome copper necessary for control of intracellular pathogen replication.