Abstract
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 (IFNγ) elicits a variety of anti-Toxoplasma activities in macrophages. Using a genome-wide CRISPR screen we identify 353 Toxoplasma genes that determine parasite fitness in naїve or IFNγ-activated murine macrophages, seven of which are further confirmed. We show that 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 the host cytoplasm. Parasites lacking GRA45 are more susceptible to IFNγ-mediated growth inhibition and have reduced virulence in mice. Together, we identify and characterize an important chaperone-like GRA in Toxoplasma and provide a resource for the community to further explore the function of Toxoplasma genes that determine fitness in IFNγ-activated macrophages.
Highlights
Macrophages play an essential role in the early immune response against Toxoplasma and are the cell type preferentially infected by the parasite in vivo
Such stimulated macrophages are extremely effective in inhibiting Toxoplasma growth—even of virulent strains like the type I (RH) strain—which would likely create a bottleneck during the selection of the mutant parasite pool
To confirm that transmembrane domain-containing GRAs were affected in Δgra[45] parasites, we examined the localization of several GRAs after exocytosis of the dense granules based on their membrane association properties: GRA5, is a PV membrane (PVM)-integrated protein with its N-terminus faces to the host cytosol;[74,75] and GRA7, associates with IVN and PVM72,73, and strands extending from PVM into the host cytosol;[76] MAF1, integrates into the PVM with its C-terminus exposed to host mitochondria[77]
Summary
Macrophages play an essential role in the early immune response against Toxoplasma and are the cell type preferentially infected by the parasite in vivo. We identify and characterize an important chaperone-like GRA in Toxoplasma and provide a resource for the community to further explore the function of Toxoplasma genes that determine fitness in IFNγ-activated macrophages. The cytokine interferon gamma (IFNγ) is essential for the control of Toxoplasma replication in host cells[4]. IFNγ-induced upregulation of immunity-related GTPases (IRGs) and guanylate binding proteins (GBPs)[6,7,8] are key mechanisms for controlling Toxoplasma in mice[9,10,11,12,13]. Toxoplasma activities in murine macrophages is parasite-straindependent, with the clonal type II and III strains being more susceptible to IFNγ-induced growth restriction compared to type I strains[21]
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