Interferon-mediated control mechanisms in human cells

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Abstract Toxoplasma gondii is a widespread parasite of animals that causes zoonotic infection in humans. Although acute infections in healthy adults are normally resolved without serious consequences, infection of immunocompromised individuals can lead to serious complications. Toxoplasma gondii is an obligate intracellular parasite, capable of infecting all nucleated cells in the body, including monocytes and macrophages. Control of infection relies on production of IFN-γ, which is essential to upregulate antimicrobial pathways in both hematopoietic and non-hematopoietic cells. Interferons activate STAT transcription factors to upregulate interferon stimulated genes (ISGs), many of which participate in antimicrobial defenses. Compared to studies in the murine system, our knowledge of how these pathways control T. gondii replication in human cells is relatively limited. Treatment of human cells with type I (IFN-β) or type II (IFN-γ) interferon leads to control of T. gondii replication. A variety of different control pathways have been implicated including nutrient limitation, guanylate binding proteins, autophagy, and production of reactive oxygen or nitrogen species. However prior studies have only focused on a few of the components that operate in each pathway, limiting our knowledge of their relative importance. To provide a comprehensive analysis of ISGs that are required for IFN- mediated control of T. gondii, we have developed a FACS-based screen to monitor parasite replication in different human cell types. Genome wide CRISPR/Cas9 screens will be employed to identify genes whose loss results in an inability to control T. gondii replication in IFN-treated cells. In parallel, we will use a lentiviral over- expression strategy to screen libraries of type I and type II ISGs to identify effectors that induce control of parasite replication. Collectively these approaches should define the repertoire of human genes that are necessary and sufficient to control intracellular parasites. Understanding such mechanisms may be important in overcoming the resistance to clearance seen in chronic toxoplasmosis and may also be important in control of other intracellular pathogens.