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 againstToxoplasmaand are the cell type preferentially infected by the parasitein vivo. Interferon gamma (IFNγ) elicits a variety of anti-Toxoplasmaactivities in macrophages. Using a genome-wide CRISPR screen we identified ∼130Toxoplasmagenes that determine parasite fitness in naїve macrophages and ∼466 genes that determine fitness in IFNγ-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 lackingGRA45mislocalize GRA effectors upon secretion, are more susceptible to IFNγ-mediated growth inhibition, and have reduced virulence in mice. Our results provide a resource for the community to further explore the function ofToxoplasmagenes that determine fitness in IFNγ-stimulated macrophages.IMPORTANCEThe intracellular parasiteToxoplasma gondiican cause congenital infections and severe disease in immunocompromised patients. The cytokine IFNγ can block parasite replication by upregulating a variety of toxoplasmacidal mechanisms in many cells, including macrophages.Toxoplasmapreferentially infects macrophages. Therefore, the parasite has evolved mechanisms to survive in these cells in the presence of IFNγ. Here, we generated pools ofToxoplasmamutants for every gene and determined which mutants were specifically depleted in IFNγ-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 IFNγ-mediated growth inhibition, and are avirulent in mice. Thus, our screen provides a resource to theToxoplasmacommunity to determine the function ofToxoplasmagenes that affect its fitness in IFNγ-stimulated macrophages.