A genome-wide loss-of-function screen identifies Toxoplasma gondii genes that determine fitness in interferon gamma-activated murine macrophages

Macrophages play an essential role in the early immune response against Toxoplasma and are the cell type preferentially infected by the parasite in vivo. Interferon gamma (IFN{gamma}) elicits a variety of anti-Toxoplasma activities in macrophages. Using a genome-wide CRISPR screen we identified [~]130 Toxoplasma genes that determine parasite fitness in naive macrophages and [~]466 genes that determine fitness in IFN{gamma}-stimulated murine macrophages, seven of which we investigated and confirmed. We show that one of these genes encodes dense granule protein GRA45, which contains a putative chaperone-like domain, and which we show is critical in preventing other GRA effectors from aggregating. Parasites lacking GRA45 mislocalize GRA effectors upon secretion, are more susceptible to IFN{gamma}-mediated growth inhibition, and have reduced virulence in mice. Our results provide a resource for the community to further explore the function of Toxoplasma genes that determine fitness in IFN{gamma}-stimulated macrophages. IMPORTANCEThe intracellular parasite Toxoplasma gondii can cause congenital infections and severe disease in immunocompromised patients. The cytokine IFN{gamma} can block parasite replication by upregulating a variety of toxoplasmacidal mechanisms in many cells, including macrophages. Toxoplasma preferentially infects macrophages. Therefore, the parasite has evolved mechanisms to survive in these cells in the presence of IFN{gamma}. Here, we generated pools of Toxoplasma mutants for every gene and determined which mutants were specifically depleted in IFN{gamma}-stimulated macrophages, thus identifying parasite genes determining fitness in these cells. We show that one of these genes encodes for a dense granule protein (GRA45) that plays an important role in preventing other GRA effectors from aggregating. Parasites without GRA45 mislocalize GRA effectors upon secretion, have enhanced susceptibility to IFN{gamma}-mediated growth inhibition, and are avirulent in mice. Thus, our screen provides a resource to the Toxoplasma community to determine the function of Toxoplasma genes that affect its fitness in IFN{gamma}-stimulated macrophages.