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A Genome-Wide Loss-Of-Function Screen Identifies Toxoplasma Gondii Genes That Determine Fitness in Interferon Gamma-Activated Mu

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A Genome-Wide Loss-Of-Function Screen Identifies Toxoplasma Gondii Genes That Determine Fitness in Interferon Gamma-Activated Mu bioRxiv preprint doi: https://doi.org/10.1101/867705; this version posted December 6, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. A genome-wide loss-of-function screen identifies Toxoplasma gondii genes that determine fitness in interferon gamma-activated murine macrophages Yifan Wang1,a, Lamba Omar Sangaré1,a, Tatiana C. Paredes-Santos1, Shruthi Krishnamurthy1, Musa A. Hassan2,3,4, Anna M. Furuta1, Benedikt M. Markus5,6, Sebastian Lourido5,7, Jeroen P.J. Saeij1,# 1 Department of Pathology, Microbiology & Immunology, School of Veterinary Medicine, University of California, Davis, Davis, California, USA. 2 College of Medicine and Veterinary Medicine, The University of Edinburgh, Edinburgh, UK. 3 The Roslin Institute, The University of Edinburgh, Edinburgh, UK. 4 Center for Tropical Livestock Health and Genetics, The University of Edinburgh, Edinburgh, UK. 5 Whitehead Institute for Biomedical Research, Cambridge, Massachusetts, USA 6 University of Freiburg, Faculty of Biology, Freiburg, Germany 7 Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA Running title: Screen in IFNγ-stimulated macrophages identifies Toxoplasma virulence genes a Y.W. and LO.S. contributed equally to this work # Address correspondence to Jeroen P.J. Saeij, [email protected] bioRxiv preprint doi: https://doi.org/10.1101/867705; this version posted December 6, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 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 ~130 Toxoplasma 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 IFNγ- stimulated macrophages. KEYWORDS: Toxoplasma, interferon gamma, CRISPR screen, effector translocation, dense granule, chaperone, macrophage bioRxiv preprint doi: https://doi.org/10.1101/867705; this version posted December 6, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. IMPORTANCE The 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 to IFNγ-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 IFNγ- stimulated macrophages. bioRxiv preprint doi: https://doi.org/10.1101/867705; this version posted December 6, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 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 to Toxoplasma demonstrating the crucial role of this cell type in IFNγ-mediated control of Toxoplasma (5). IFNγ-induced upregulation of immunity-related GTPases (IRGs) and guanylate binding proteins (GBPs) (6–8) are key mechanisms for controlling Toxoplasma in mice (9–13). IRGs and GBPs can bind and subsequently vesiculate the PVM, eventually leading to the death of the parasite (14–17). In addition, IFNγ induces the production of nitric oxide (NO) and reactive oxygen species, which lead to parasite damage and growth restriction in murine macrophages (18). However, the potency of IFNγ-induced toxoplasmacidal activities in murine macrophages is parasite-strain dependent, with the clonal type 2 and 3 strains being more susceptible to IFNγ-induced growth restriction compared to type 1 strains (19). Toxoplasma 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 in (20)). ROP18 (encoding a secreted kinase) (21, 22) and ROP5 (encoding a member of an expanded family of pseudokinases) (23, 24) are highly polymorphic and, together with ROP17 (25) and GRA7 (26, 27), account for differences in parasite virulence in mice by cooperatively blocking IRG (14, 28–30) and GBP (6, 31) loading on the PVM. Other parasite strain-specific effectors include ROP16 and GRA15, which can affect GBP loading on the PVM (32) and macrophage polarization into the classical (M1) or bioRxiv preprint doi: https://doi.org/10.1101/867705; this version posted December 6, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. alternative (M2) activation phenotypes (33). The pseudokinase ROP54 also inhibits GBP loading on the PVM (34). Additional secreted effectors, such as GRA12, affect Toxoplasma survival in murine macrophages without affecting the loading of IRGs/GBPs to the PVM (35). Besides targeting IFNγ- induced host cell effectors, Toxoplasma can directly inhibit IFNγ signaling. For example, through a GRA (TgIST) secreted beyond the PVM (36, 37), Toxoplasma can directly inhibit STAT1 transcriptional activity (38–40), an essential component of the IFNγ signaling pathway (41). The secretion of GRAs beyond the PVM into the host cell cytosol is dependent on the Toxoplasma proteins MYR1/2/3 (42, 43), which might form a PVM-localized translocon. A Golgi-resident aspartyl protease ASP5, which cleaves the Toxoplasma export element (TEXEL, RRLxx) motif in some GRAs, is required for exported GRAs to reach the host cytosol and also for the correct localization of other GRAs confined to the PV (44–46). Many of the above-mentioned Toxoplasma virulence genes were identified using genetic crosses between strains that differ in virulence in mice. However, this approach fails to identify Toxoplasma genes that determine strain-independent survival in IFNγ-activated macrophages. Here we used a genome-wide loss-of-function screen in the type 1 (RH) strain, which is resistant to IRG/GBP-mediated killing, to identify ~466 Toxoplasma genes that determine fitness in IFNγ-activated murine macrophages and ~130 genes that determine fitness in naїve macrophages. Seven of the top hits, including GRA45, were confirmed by generating and testing single gene knockouts in functional parasite growth assays in IFNγ-activated murine macrophages. We show that GRA45 plays an important role in preventing aggregation of other GRA effectors in the secretory pathway. Δgra45 parasites have enhanced susceptibility to IFNγ-mediated growth inhibition in murine, rat, and human macrophages and significantly reduced virulence in mice. Our results provide a resource to the community to investigate the mechanism by which other Toxoplasma genes affect fitness in naїve or activated macrophages. RESULTS: bioRxiv preprint doi: https://doi.org/10.1101/867705; this version posted December 6, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. A loss-of-function screen identifies Toxoplasma genes that determine fitness in naïve and IFNγ- activated murine macrophages. We previously identified Toxoplasma genes that determine
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