The percentage of vacuoles with only PV-lumen staining was quantified as shown on the right. the variety of parasite lines described in this manuscript) and custom code used in the analysis of CRISPR screen data are Rabbit polyclonal to ACE2 available from the corresponding author (contact: Jeroen P.J. Saeij, firstname.lastname@example.org) upon reasonable request.?Source data are provided with this paper. Abstract Macrophages play an essential role in the early immune response against and are the cell type preferentially infected by the parasite in vivo. Interferon gamma (IFN) elicits a variety of anti-activities in macrophages. Using a genome-wide CRISPR screen we identify 353 genes that determine parasite fitness in na?ve or IFN-activated murine macrophages, seven of which are further confirmed. We show that Dot1L-IN-1 one of these genes encodes dense granule protein GRA45, which has a chaperone-like domain, is critical for correct localization of GRAs into the PVM and secretion of GRA effectors into Dot1L-IN-1 the host cytoplasm. Parasites lacking GRA45 are more susceptible to IFN-mediated growth inhibition and have reduced virulence in mice. Together, we identify and characterize an important chaperone-like GRA in and Dot1L-IN-1 provide a resource for the community to further explore the function of genes that determine fitness in IFN-activated macrophages. is an obligate intracellular parasite that causes disease in immunocompromised individuals, such as HIV/AIDS patients, and when contracted congenitally1. It causes lifelong infections by converting from rapidly dividing tachyzoite stages into encysted slow-growing bradyzoites, which mainly localize to the brain and muscle tissues. Once established in the host cell, replication in host cells4. Mice with macrophages that can no longer respond to IFN are extremely susceptible to demonstrating their crucial role in IFN-mediated control of in mice9C13. IRGs and GBPs can bind and vesiculate the PVM, eventually leading to the death of the parasite14C17. In addition, IFN induces the production of nitric oxide (NO) and reactive oxygen species (ROS), which lead to parasite damage and growth restriction in murine macrophages18. IFN stimulation also changes the metabolism of cells thereby affecting the availability of nutrients to pathogens19. For example, IFN-activated cells can limit the availability of iron, L-tryptophan, cholesterol, polyamines, and dNTPs20. The potency of IFN-induced anti-activities in murine macrophages is parasite-strain-dependent, with the clonal type II and III strains being more susceptible to IFN-induced growth restriction compared to type I strains21. co-opts host cells by secreting ROP and GRA effector proteins, from organelles called rhoptries and dense granules, respectively, into the host cell or onto the PVM (reviewed in22). (encoding a secreted kinase)23,24, and (encoding a member of an expanded family of pseudokinases)25,26 are highly polymorphic and, together with ROP1727 and GRA728,29, account for differences in parasite virulence in mice by cooperatively blocking IRGs14,30C32 and GBPs6,33 loading on the PVM. Other parasite-strain-specific effectors include ROP16 and GRA15, which can affect GBP1 loading on the PVM34 and macrophage polarization into the classical (M1) or alternative (M2) activation phenotypes35. GRA15 was recently shown to recruit IRGB6 Dot1L-IN-1 and GBP1-5 to the PVM via interacting with TRAF6, thereby enhancing the susceptibility to IFN-induced parasite elimination36. The pseudokinase ROP54 also inhibits GBP2 loading on the PVM37. Additional secreted effectors, such as GRA12, affect survival in murine macrophages without affecting the loading of IRGB6 to the PVM38. Besides targeting IFN-induced host cell effectors, can directly inhibit IFN signaling. For example, through a GRA (can directly inhibit STAT1 transcriptional activity41C43, an essential component of the IFN signaling pathway44. The secretion of GRAs beyond the PVM into the host cell cytosol is dependent on the MYR complex45C47, which might form a PVM-localized translocon. A Golgi-resident aspartyl protease ASP5, which cleaves the export element (TEXEL, RRLxx) motif in some GRAs,.