A genome-wide loss-of-function screen identifiesToxoplasma gondiigenes that determine fitness in interferon gamma-activated murine macrophages

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 identified ~130Toxoplasma genes 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 lacking GRA45 mislocalize 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 of Toxoplasma genes that determine fitness in IFNγstimulated macrophages. IMPORTANCEThe intracellular parasite Toxoplasma gondii can 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. Toxoplasma preferentially infects macrophages. Therefore, the parasite has evolved mechanisms to survive in these cells in the presence of IFNγ. Here, we generated pools of Toxoplasma mutants 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 toIFNγ-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 IFNγstimulated macrophages. INTRODUCTION:Toxoplasma gondii is an obligate intracellular parasite that causes disease in immunocompromised individuals, such as HIV/AIDS patients, and when contracted congenitally (1). 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, Toxoplasma resides in a unique replication niche called the parasitophorous vacuole (PV), which is separated from the host cytoplasm by the PV membrane (PVM) and does not fuse with the endolysosomal system (2,3).The cytokine interferon gamma (IFNγ) is essential for the control of Toxoplasma replication in host cells (4). Mice with macrophages that can no longer respond to IFNγ are extremely susceptible toToxoplasma demonstrating the crucial role of this cell type in IFNγ-mediated control of Toxoplasma (5).IFNγ-induced upregulation of im...

Toxoplasma GRA 15 limits parasite growth in IFN γ‐activated fibroblasts through TRAF ubiquitin ligases

“…It is likely that in human cells, other parasite virulence factors, besides GRA15, likely determine susceptibility to IFNγ‐mediated elimination. A systematic identification of such factors could be possible through genetic screens using CRISPR‐Cas9 approaches (Wang et al , 2019c).…”

“…A genetically engineered RH strain expressing type II GRA15, PruΔ gra15 , and PruΔ gra15 + GRA15‐HA (GRA15‐complemented strain) was described previously (Rosowski et al , ). Toxoplasma gondii RH strains endotagged with GRA43‐HA and GRA45‐HA were made as described previously (Wang et al , 2019b; Wang et al , 2019c).…”

Toxoplasma gondiiPPM3C, a secreted protein phosphatase, affects parasitophorous vacuole effector export

“…Precedence for this notion can be found in the P granules of C. elegans embryos, in which phosphorylation of intrinsically disordered proteins drives de-formation of phase separated granules whereas phosphatase activity induces the opposite effect [46], although an obvious difference in this system is that P granule formation involves RNA as a seed for intrinsically disordered proteins with RNA binding motifs to form a phase separated state. The GRA45 protein was recently shown to facilitate effector protein export by seemingly preventing the aggregation of vacuolar GRA16 and GRA24 [44], which provides support for the notion that effector proteins can aggregate within the parasitophorous vacuole, potentially due to their predicted disordered state. Based on the impaired export of phosphomimetic GRA16, we propose that the de-phosphorylation of GRA16 and GRA28 somehow enables these effectors to traverse the hypothetical vacuolar protein translocon more efficiently.…”

“…Exported effectors such as GRA16 and TgIST have been elegantly characterized and shown to influence host cell signaling in profound ways that benefit parasite intracellular development [13, 17, 18]. Several effectors exported for the parasitophorous vacuole have been discovered apart from GRA16, GRA28, and TgIST [38–41], while the list of proteins necessary for the export of these proteins has also been growing [16, 42–44], including an acid phosphatase domain containing protein, GRA44. Our data demonstrate that although TgPPM3C is not essential for effector export, this phosphatase likely optimizes effector export based on the deficiencies observed in GRA16 and GRA28 export and host c-Myc induction (Fig 4A-B).…”

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“…Exported effectors such as GRA16 and TgIST have been elegantly characterized and shown to influence host cell signaling in profound ways that benefit the intracellular development of the parasite [23,26,27]. Several effectors exported from the parasitophorous vacuole have been described apart from those explored in this study [9,28,29,46], while the list of proteins necessary for the export of these proteins has also been growing, including an acid phosphatase domain-containing protein in GRA44 [21,[47][48][49]. Cygan et.…”