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A heterotrimeric complex of Toxoplasma proteins promotes parasite survival in interferon gamma-stimulated human cells [1]

['Eloise J. Lockyer', 'Signalling In Apicomplexan Parasites Laboratory', 'The Francis Crick Institute', 'London', 'United Kingdom', 'Francesca Torelli', 'Simon Butterworth', 'Ok-Ryul Song', 'High-Throughput Screening Science Technology Platform', 'Steven Howell']

Date: 2023-07

Abstract Toxoplasma gondii secretes protein effectors to subvert the human immune system sufficiently to establish a chronic infection. Relative to murine infections, little is known about which parasite effectors disarm human immune responses. Here, we used targeted CRISPR screening to identify secreted protein effectors required for parasite survival in IFNγ-activated human cells. Independent screens were carried out using 2 Toxoplasma strains that differ in virulence in mice, leading to the identification of effectors required for survival in IFNγ-activated human cells. We identify the secreted protein GRA57 and 2 other proteins, GRA70 and GRA71, that together form a complex which enhances the ability of parasites to persist in IFNγ-activated human foreskin fibroblasts (HFFs). Components of the protein machinery required for export of Toxoplasma proteins into the host cell were also found to be important for parasite resistance to IFNγ in human cells, but these export components function independently of the identified protein complex. Host-mediated ubiquitination of the parasite vacuole has previously been associated with increased parasite clearance from human cells, but we find that vacuoles from GRA57, GRA70, and GRA71 knockout strains are surprisingly less ubiquitinated by the host cell. We hypothesise that this is likely a secondary consequence of deletion of the complex, unlinked to the IFNγ resistance mediated by these effectors.

Citation: Lockyer EJ, Torelli F, Butterworth S, Song O-R, Howell S, Weston A, et al. (2023) A heterotrimeric complex of Toxoplasma proteins promotes parasite survival in interferon gamma-stimulated human cells. PLoS Biol 21(7): e3002202. https://doi.org/10.1371/journal.pbio.3002202 Academic Editor: Kami Kim, University of South Florida, UNITED STATES Received: April 7, 2023; Accepted: June 16, 2023; Published: July 17, 2023 Copyright: © 2023 Lockyer et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: All relevant data are within the paper and its Supporting information files. Proteomics raw data are uploaded into the PRIDE repository (PXD041352), mRNAseq raw data files have been submitted to GEO with the accession number (GSE230866). Funding: The work of EJL, FT, SB and MT was supported by the Francis Crick Institute which receives its core funding from Cancer Research UK (CC2133), the UK Medical Research Council (CC2133), and the Wellcome Trust (CC2133). FT received funding from the Deutsche Forschungsgemeinschaft (TO 1349/1-1). OS, SH, AW and PE were supported by the Science Technology Platforms at the Francis Crick Institute, which receive funding from Cancer Research UK (CC0199), the UK Medical Research Council (CC0199), and the Wellcome Trust (CC0199). The funders play no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. Abbreviations: BMDM, bone marrow-derived macrophage; BSA, bovine serum albumin; FDR, false detection rate; GBP1, guanylate-binding protein 1; gDNA, genomic DNA; GEO, Gene Expression Omnibus; HFF, human foreskin fibroblast; HUVEC, human umbilical vein endothelial cell; IRG, immunity-related GTPase; ISG, IFN-stimulated gene; LC, liquid chromatography; LFQ, label-free quantification; MAD, median absolute deviation; MEF, mouse embryonic fibroblast; PFA, paraformaldehyde; PV, parasitophorous vacuole; PVM, parasitophorous vacuole membrane; sgRNA, single- guide RNA; TEM, transmission electron microscopy; TFA, trifluoroacetic acid; TMD, transmembrane domain

Introduction Toxoplasma gondii is an obligate intracellular parasite that can infect any nucleated cell of virtually any warm-blooded animal. With an estimated human worldwide seroprevalence of 30%, it is also likely one of the most prevalent human protozoan parasites [1]. Though Toxoplasma can cause disease in immunocompromised patients or following congenital transmission, most immunocompetent individuals control the infection through the combined action of the adaptive and innate immune systems. The Toxoplasma gondii lifecycle comprises 2 distinct phases, a sexual stage that takes place exclusively in the definitive feline host and an asexual stage that can occur in a very broad range of intermediate hosts [2]. The evolutionary importance of an intermediate host is intrinsically linked to the frequency with which it promotes transmission of Toxoplasma parasites back to the felid definitive host [3]. Rodent species and other small warm-blooded animals that are prey to felines are therefore important natural intermediate hosts for Toxoplasma, but as humans are very rarely natural feline prey, they are considered “accidental” intermediate hosts. Once within an intermediate host, Toxoplasma actively invades host cells and resides in a host-derived membrane-bound vacuole known as the parasitophorous vacuole (PV). In mice and humans, host cell-autonomous immunity to Toxoplasma is mediated by the type II interferon IFNγ. IFNγ stimulation leads to the up-regulation of hundreds of IFN-stimulated genes (ISGs) through STAT1-induced transcription of gamma-activated sequence elements [4,5]. To counteract the IFNγ response, Toxoplasma secretes protein effectors from specialised rhoptry and dense granule organelles. Dense granule proteins (GRAs) and rhoptry proteins (ROPs) transform the host cell by altering gene transcription and host metabolism, mediating the acquisition of nutrients, removing host cell factors from the PV membrane (PVM) and targeting proinflammatory signalling pathways (reviewed in [6–11]). Global mapping of the Toxoplasma proteome using hyperplexed localisation of organelle proteins by isotope tagging (hyperLOPIT) has predicted the existence of at least 124 GRAs and 106 ROPs [12]. Sequence polymorphisms and differential expression of secreted effectors across the 3 main clonal lineages of Toxoplasma (types I–III) have been shown to determine differences in strain virulence [13,14]; however, the vast majority of ROPs and GRAs remain uncharacterised. GRAs can be translocated across the parasite plasma membrane and PVM into the host cell cytoplasm via the MYR complex, a multi-protein putative translocon. To date, 8 proteins have been identified as necessary for protein translocation: the putative MYR translocon components; MYR1, MYR2, MYR3, MYR4, and ROP17 [15–18], as well as GRA44, GRA45, and ASP5 [18,19]. The secretion of MYR-dependent effectors is responsible for the vast majority of host cell transcriptome changes induced by Toxoplasma infection in both human and mouse cells [20,21]. MYR1 knockouts display an expected reduction in virulence in vivo, but this phenotype is lost in pooled knockout CRISPR screens, suggesting MYR1-dependent effectors may exert paracrine effects at the site of infection [22]. How the MYR proteins directly facilitate protein translocation is not yet understood, and while MYR1 and MYR3 stably associate in vitro [16], it is not yet clear whether all MYRs form a single complex required for protein translocation. Efforts to characterise the function of Toxoplasma-secreted effectors historically centred on their role in the murine host, leading to the discovery of ROP18, ROP5, and ROP17 [23–26]. In mice, these effectors act cooperatively to prevent the loading of the immunity-related GTPase (IRG) family of large GTPases [27–32], which are strongly induced by IFNγ in multiple mouse cell types [33,34]. IRG proteins load sequentially onto the PVM and cooperatively oligomerise to vesiculate and rupture the membrane, leading to parasite clearance and necrotic host cell death [33,35,36]. The allelic combination of ROP18/ROP5/ROP17 in each parasite strain determines the strain-specific virulence of Toxoplasma in mice [37]. While mice have a dramatically expanded family of 23 IRGs, IRGs are largely absent in humans having been mostly lost in the primate lineage prior to the evolution of monkeys [38]. As a result, ROP18/ROP17/ROP5 do not function similarly to resist vacuole clearance in human cells [31]. In humans, the IFNγ-induced responses to Toxoplasma are highly cell-type specific. These mechanisms include autophagic clearance [39], endo-lysosomal destruction [40,41], nutrient deprivation [42,43], or induction of host cell death [44]. In human macrophages, the p65 GTPase guanylate-binding protein 1 (GBP1) has been demonstrated to mediate killing of Toxoplasma through recruitment to and disruption of the PVM, leading to host cell apoptosis [45]. In multiple mouse and human cell types, IFNγ-driven ubiquitination of the PVM by host E3 ligases serves as an initial marker for eventual parasite clearance. In mouse embryonic fibroblasts (MEFs) and bone marrow-derived macrophages (BMDMs), TRAF6-mediated ubiquitination of types II and III vacuoles enhances the recruitment of GBPs, resulting in rupture of the PVM [41,46]. The E3 ligase TRIM21 has also been shown to promote clearance of types II and III strains in vivo [47] and ubiquitinates type III vacuoles in human foreskin fibroblasts (HFFs) resulting in parasite growth restriction [48]. In human umbilical vein endothelial cells (HUVECs), K63-linked ubiquitination initiates a cascade of autophagy marker recruitment that culminates in acidification of the parasite vacuole [40], while in HeLa cells ubiquitination instead leads to recruitment of LC3 and stunting of parasite growth [39]. Compared to types II and III parasites, type I parasites are more resistant to ubiquitination and clearance in both HUVECs and HeLa cells. Recent evidence suggests a major function for the E3 ligase RNF213 in ubiquitination of the PVM and enhancing parasite clearance, in multiple human cell types and in a strain-independent manner [49,50]. The importance of each E3 ligase and ubiquitin linkage therefore depends on the exact combination of host species, parasite strain, and cell type studied, but nevertheless ubiquitination has emerged as a key process in the regulation of Toxoplasma clearance. The Toxoplasma-derived targets that are recognised by these host E3 ligases remain to be determined. Toxoplasma effectors that have been described to counteract IFNγ in human cells include the host transcriptional repressor IST, which blocks IFNγ-stimulated gene transcription [51,52]. However, IST can only prevent clearance in cells that have not been pre-stimulated with IFNγ, a condition that may only be met in the early stages of infection. Another effector is NSM, which blocks host cell necroptosis during the bradyzoite stages of infection [53]. More recently, we have shown that ROP1 counteracts IFNγ immune responses in both murine and human macrophages [54]. Finally, in human THP-1-derived macrophages, deletion of the chaperone protein GRA45 increases the sensitivity of parasites to IFNγ-mediated growth inhibition [55]. There therefore remains a significant gap in our understanding of which effectors mediate Toxoplasma virulence in humans [3,56]. To address this gap, we performed targeted CRISPR screening of the Toxoplasma “secretome” [12] during infection of unstimulated and IFNγ-stimulated HFFs, using our previously described CRISPR platform [22]. Independent experiments were performed in type I (RH) and type II (PRU) parasite strains, which allowed identification of both strain-dependent and independent effectors. We found that GRA57 is a strain-independent effector that protects Toxoplasma from IFNγ-mediated vacuole clearance in HFFs. GRA57 was found to interact with 2 other dense granule proteins, GRA70 (TGME49_249990) and GRA71 (TGME49_309600), which resist IFNγ-mediated vacuole clearance to a similar degree as GRA57, indicating that the 3 proteins function in the same pathway, possibly as a complex. Deletion of any member of this trio results in reduced PVM ubiquitination in HFFs. We also found 2 components of the MYR translocon, MYR1 and MYR3, contribute to IFNγ resistance in HFFs, though we expect that one or several unidentified MYR-dependent effectors play an additional significant role. As GRA57 does not impact GRA export, and MYR1 and MYR3 do not display an ubiquitination phenotype, we conclude that GRA57 and the MYR proteins function independently of each other. These data suggest that a novel MYR-independent trimeric complex of dense granule proteins localised within the PV contribute to resisting IFNγ-induced vacuole clearance in HFFs.

Discussion In this study, we used targeted CRISPR screens to identify Toxoplasma-secreted virulence factors that protect the parasite from human cell-autonomous immunity. CRISPR screens were carried out in RH and PRU Toxoplasma strains, revealing a novel complex of 3 interacting dense granule effectors, comprising GRA57, GRA70, and GRA71, that contribute equally to Toxoplasma resistance to IFNγ-induced responses in HFFs. In addition to GRA57 and its 2 partner proteins, we show that 2 components of the MYR effector export machinery, MYR1 and MYR3, are important for parasite survival in IFNγ-activated HFFs in both pooled and single knockout infections. Other MYR components including the kinase ROP17 [17] displayed an IFNγ-survival phenotype in both screens, whereas the effector chaperone GRA45 [55] was essential for IFNγ survival in only the PRU screen. Together, this would suggest that the GRA protein translocation machinery of Toxoplasma is required for survival in IFNγ-treated HFFs. However, MYR2 and MYR4 displayed no phenotype in either of our screens, though it is possible these are false-negative results. Only MYR1 and MYR3 have previously been shown to stably associate with each other in vitro [16], therefore, whether MYR1 and MYR3 have an important additional function in HFFs that is distinct from effector export would be interesting to explore in the future. In contrast, neither ROP18 nor GRA12 had a major phenotype in IFNγ-stimulated HFFs, whereas in murine screens ROP18 and GRA12 consistently emerge as the most important secreted effectors for parasite survival [22,54,55]. This emphasises the importance of using human in vitro systems to identify parasite effectors that mediate the pathogenesis of Toxoplasma in the human host. In both the RH and PRU screens, we observed high levels of parasite restriction in HFFs, with approximately 90% of the mutant population restricted in cells pre-stimulated with 5 U/ml IFNγ, a dose 20-fold less than that commonly used in literature. HFFs are extensively used for the continuous culture of Toxoplasma and are often considered as “non-immune” cells, but these results support that structural cells can be important components of the innate immune response to Toxoplasma infection [73]. Other human non-hematopoietic cells have also been demonstrated to restrict Toxoplasma in response to IFNγ signalling, including neurons [74], epithelial [39] and endothelial cells [40]. High-content imaging of parasite restriction in HFFs revealed the RHΔMYR1 and RHΔMYR3 strains are highly susceptible to restriction through both increased vacuole clearance and growth restriction. Given the pleiotropic effects on the host that are mediated by MYR-dependent effector export [21], we assume that the abrogation of all effector export leads to hypersensitivity to multiple IFNγ-induced restriction pathways in HFFs. This is further supported as no other dense granule protein with an equally strong phenotype was identified in our screen, although it is possible that targets were filtered out due to variability between guides. As our library is constructed from hyperLOPIT prediction [12], it could also be possible that some GRAs or ROPs are omitted if not previously annotated as localised to the dense granules or rhoptries. In contrast to MYR1 and MYR3 knockouts, single knockouts of GRA57, GRA70, or GRA71 were more sensitive to IFNγ-mediated vacuole clearance but did not display decreased replication in activated cells. As GRA57 was not shown to influence effector export, and both GRA57 and GRA70 do not appear to localise outside of the PV, we assume that this dense granule complex is not exported via the MYR translocon and functions independently of the MYR complex to promote parasite survival in IFNγ-activated cells. In contrast to HFFs, we observed no significant impact on IFNγ survival in MEFs upon deletion of GRA57, with no previously identified phenotype for GRA57/GRA70/GRA71 in murine screens [22,54,55,64]. From this, we infer that the trimeric complex functions to subvert an IFNγ-induced response that is present in HFFs but absent or less dominant in MEFs. One such response is the induction of HFF cell death and concomitant premature parasite egress [44]. While this manuscript was under review, another group found a role for GRA57, GRA70, and GRA71 in preventing premature egress in IFNγ-stimulated HFFs [71]. We observed a larger IFNγ-dependent decrease in vacuole numbers when HFFs were infected with knockouts of the GRA57/GRA70/GRA71 complex, an effect which could be explained by early egress of these strains in response to IFNγ. The mechanism through which IFNγ induces early egress however remains unknown. It has previously been shown that endolysosomal acidification of the PV mediates clearance of PRU parasites in IFNγ-activated HFFs, an effect that is inhibited through unknown mechanisms in RH parasites [41]. Acidification of the PV during the normal lytic cycle of Toxoplasma serves as a signal for microneme secretion and parasite egress [75]. Therefore, acidification induced by host cell endolysosomal fusion with the PV may induce premature parasite egress, thereby limiting parasite replication and dissemination. Future work will aim to determine if the GRA57/GRA70/GRA71 complex functions to resist IFNγ-induced vacuole acidification in HFFs. Autophagy has emerged as a major clearance mechanism for Toxoplasma [76] and multiple other intracellular pathogens [77]. In our efforts to determine if host autophagy contributed to the increased clearance of GRA57/GRA70/GRA71 knockouts, we unexpectedly found a marked reduction in the percent of these knockout vacuoles targeted by host ubiquitin in HFFs and MEFs, in both the presence and absence of IFNγ-stimulation. For GRA57 knockouts, we determined there was specific depletion of K63- and M1-linked ubiquitin chains at the PVM, correlating with reduced recruitment of the host E3 ligase RNF213, which has been demonstrated to facilitate attachment of these ubiquitin linkages to the PVM [49]. We believe there is unlikely to be a causal link between ubiquitin recruitment and the increased restriction of GRA57/GRA70/GRA71 knockouts in IFNγ-activated HFFs, given that (a) reduced ubiquitin recruitment was also observed in MEFs, in which the knockouts strains are not as susceptible to IFNγ; (b) reduced RNF213 and ubiquitin recruitment was also evident in unstimulated cells; and (c) a decrease in vacuole ubiquitination should correlate with an increase in parasite survival. Mukhopadhyay and colleagues [41] have shown that ubiquitination of Toxoplasma vacuoles in HFFs is not intrinsically linked to downstream fusion with the endolysosomal system, supporting that our observed decrease in vacuole ubiquitination may be a secondary effect of deletion of the GRA57/GRA70/GRA71 complex. At 3 h postinfection, the total percentage of vacuoles with ubiquitin recruitment is reduced in these knockouts, but not entirely ablated (Fig 5B and 5D). Therefore, an alternative possibility is that in the absence of the complex, the vacuoles become hypersensitive to the remaining ubiquitination and subsequent disruption. Recent work has found that RNF213, an IFNγ-induced host E3 ubiquitin ligase, mediates the majority of ubiquitin recruitment to both RH and PRU Toxoplasma vacuoles in HFFs [49,50]; however, the target of this ligase is currently unknown. The observation that GRA57 deletion in both RH and PRU Toxoplasma leads to reduced recruitment of RNF213 to the PV poses the intriguing question of whether the GRA57/GRA70/GRA71 complex is required for RNF213 recruitment, is a direct target of ubiquitination, or is responsible for correct positioning of an RNF213 target at the PV. Each of these possibilities will be interesting to follow up to better understand the function of RNF213 in human cell-autonomous restriction of Toxoplasma. In conclusion, we identify several proteins that are required for survival in HFFs under conditions of IFNγ restriction in 2 Toxoplasma strains that differ in virulence. We identify a complex of 3 proteins that is required to protect Toxoplasma parasites from killing by the host cell. While deletion of the complex components leads to reduced ubiquitination of the parasite vacuole, we suggest that functional consequences of the complex deletion likely go beyond a role in ubiquitination. Our study did not identify which host cell pathway is counteracted by the GRA57/GRA70/GRA71 complex, though we ruled out a role in effector export, transcriptional modulation, tryptophan metabolism, and vacuole ultrastructure formation. Data described by Krishnamurthy and colleagues [71] suggests a role for the GRA57/GRA70/GRA71 complex in preventing IFNγ-induced premature egress, but the host factors mediating this phenotype remain to be identified. Future work to gain mechanistic insight into this complex would ideally use an unbiased knockout or knockdown screen of host IFNγ-stimulated genes, similar to that described by Matta and colleagues [50], to identify which host pathways are counteracted by the GRA57/GRA70/GRA71 complex. This would also provide a basis for further work aimed at understanding the biochemical functions mediated by the complex. It is important to note that none of the 3 proteins identified here to protect the parasite against the IFNγ response in human cells have been identified in previous screens in murine cells or mice. Given this discrepancy, it will be interesting to determine if this complex targets a host pathway not present or active in murine cells. As humans are primarily an accidental host for Toxoplasma, it is highly likely that the GRA57 complex evolved to protect the parasite in species other than humans. Comparison of GRA57 and MYR function in cells of various origins may therefore reveal commonalities in the cell autonomous immune response between humans and other species that can be infected by Toxoplasma.

Methods CRISPR-Cas9 screens Parasite transfections. To generate a pooled population of parasite effector knockouts, we used a plasmid library comprising 1,299 single-guide RNAs (sgRNAs) targeting 253 predicted secreted proteins, with an average of 5 sgRNAs/gene, with sgRNAs integrated into a pCas9-GFP-T2A-HXGPRT::sgRNA vector. The plasmid pool was linearised overnight with KpnI-HF (NEB, R3142), purified by phenol-chloroform precipitation, then resuspended in P3 (Lonza, V4XP-3024) transfection buffer at a concentration of 1 μg/μl. A minimum of 90 million parasites (RHΔHXGPRT or PRUΔHXGPRT) were transfected in triplicate using an Amaxa 4D Nucleofector (Lonza, AAF-1003X) with pulse code EO-115, with 30 μg/transfection of the purified sgRNA plasmid library. Transfected populations were selected for after 24 h using 25 μg/mL mycophenolic acid (Sigma–Aldrich, 89287) and 50 μg/mL xanthine (Sigma–Aldrich, X3627) (M/X). Transfection efficiency was assessed via plaque assay, achieving a final guide coverage of 174X and 68X in the RH and PRU screens, respectively. Three days posttransfection, parasites were syringe lysed and added to HFF monolayers with 100 U/ml benzonase (Merck, E1014-25KU) overnight to remove traces of input DNA. Eight days posttransfection, subset parasite populations were harvested for genomic DNA (gDNA) extraction to determine guide abundance in the starting inoculum. Remaining triplicate transfections were combined to generate the inoculum used for infections. Infections. HFFs were grown to confluency in T175 flasks, then stimulated with 5 U/ml human IFNγ (Bio-Techne, 285-IF-100) for 24 h preinfection. For infections, parasites were isolated from HFFs by syringe lysis through a 30-gauge needle (×3) and passed through 5 μm filters (Millipore, SMWP04700), then added to HFFs at an MOI of 0.2 (1.4 × 106 parasites/flask) for 48 h. At 48 h postinfection, parasites were harvested as above from HFFs, counted, and then a subset were added to new flasks of IFNγ treatment-matched HFFs at an MOI of 0.2 for a further 48 h. For each round of infection surviving parasites populations were expanded in unstimulated HFFs for a further 48 h prior to harvesting for gDNA extraction. gRNA isolation and sequencing. Genomic DNA was extracted from samples using Qiagen DNEasy Blood kit, then guide sequences were amplified from gDNA and the plasmid pool by nested PCR using KAPA HIFI Hotstart PCR kit (Kapa Biosystems, KK2501), as previously described in [22]. Primers used for nested PCR are listed in S11 Data (primers 1–20). Purified PCR products were then sequenced on a HiSeq400 (Illumina) with single end 100 bp reads at a minimum read depth of 5 million reads/sample. Sequencing data analysis. gRNA sequences were aligned to a reference library as described previously in [22,54]. The lowest 1.5 percentile of guides expressed across all samples were removed from the analysis. Counts were normalised using the median of ratios, then genes represented by fewer than 3 matching guides were removed from the analysis. The median L2FC for each gene was calculated from the normalised counts at the end of growth in pre-stimulated HFFs relative to unstimulated HFFs, or from counts at the end of growth in unstimulated HFFs relative to the counts in the starting inoculum sample. The median absolute deviation (MAD) score across gRNA L2FCs targeting each gene was calculated, and genes with the highest 1.5% of MAD scores were removed from the analysis. A DISCO score based on the local FDR-corrected q-value was calculated for each L2FC to compare between untreated and IFNγ pre-stimulated HFFs. Parasite and host cell culture Toxoplasma gondii strains were maintained by serial passage every 2 to 3 days in HFFs (ATCC, SCRC-1041). For experiments, parasites were isolated by syringe lysis through a 27-gauge needle and passed through 5 μm filters. Parasite genotypes were verified by restriction fragment length polymorphism analysis of the SAG3 gene using primers 70 and 71 [78]. HFFs and MEFs (gift from Felix Randow) were maintained in DMEM with GlutaMAX (Gibco) supplemented with 10% foetal bovine serum (Gibco). Parental strains used were RHΔHXGPRT and PRUΔHXGPRT [79], RHΔKU80 [80], and PRUΔKU80 [81]. Generation of parasite cell lines Knockouts. For generation of RHΔGRA57 and PRUΔGRA57 strains, 2 guides were designed targeting exon 1 and exon 7 of the GRA57 coding sequence (CDS). Guides were integrated separately into a pCas9-GFP::sgRNA vector by inverse PCR using a general reverse primer [21] and primers 24 or 25. The exon 1 targeting plasmid was digested with XhoI/KpnI (NEB), and the exon 7 guide was amplified with primers 22 and 23 to facilitate Gibson cloning of the exon 7 guide into the same backbone. For all other knockouts generated in this study (RHΔMYR3, RHΔGRA70, and RHΔGRA71), single-guide plasmids were generated using primers 26–28. Homology repair cassettes were generated by PCR amplification from ProGRA1-mCherry-T2A-HXGPRT-TerGRA2, using primers with 40 bp flanking homology to the 5′ and 3′ UTRs of each gene (primers 29–36). A total of 10 μg of repair cassette was purified with 5 μg of pCas9-GFP::sgRNA and transfected into RHΔKu80 or PRUΔKu80 parasites, using an Amaxa 4D Nucleofector as described above. Transfectants were selected 24 h posttransfection with 25 μg/ml of mycophenolic acid (Merck) and 50 μg/ml xanthine (Sigma) (M/X) and cloned by limiting dilution. Integration of the mCherry-HXGPRT repair templates was verified by PCR using primers 37–46. Complementation. To complement the RHΔGRA57 and PRUΔGRA57 strains, the 5′ UTR and CDS of GRA57 was generated through a combination of PCR amplification from gDNA (5′ UTR, exons 1 and 7- primers 47–54) and amplification from gBlocks (IDT) for exons 2–4 and exon 6 (listed in S11 Data). PCR products were inserted into the pUPRT vector [82] by Gibson assembly. The pUPRT:GRA57-HA plasmid was linearised with ScaI, then 10 μg was transfected into RHΔGRA57 or PRUΔGRA57 alongside 1 μg of pCas9-GFP::UPRT. Transfectants were selected 24 h posttransfection with 5 μM 5′-fluo-2′-deoxyuridine (FUDR), then cloned and verified by PCR at the UPRT locus using primers 55–58. Endogenous tagging. To endogenously tag GRA57 with an HA epitope tag at the C-terminus, a pCas9-GFP::sgRNA plasmid targeting the 3′ end of exon 7 of GRA57 was generated by inverse PCR from pCas9-GFP::sgRNA using primers 21 and 59. A repair cassette with 40 bp flanking homology was amplified by PCR from HA-TerGRA2::ProDHFR-HXGPRT-TerDHFR using primers 61 and 62. Approximately 10 μg of repair cassette was purified with 5 μg of pCas9-GFP::sgRNA and transfected into RHΔKu80 as above. For endogenous tagging of GRA70 with a C-terminal V5 tag, the pCas9-GFP::sgRNA plasmid was generated using primers 21 and 60, and the repair cassette was generated by PCR from a V5-TerGRA2::ProDHFR-HXGPRT-TerDHFR construct, using primers 63 and 64. Transfectants were selected 24 h posttransfection with 25 μg/ml of mycophenolic acid (Merck) and 50 μg/ml xanthine (Sigma) (M/X), then cloned and verified by PCR using primers 65–69. All primers used for generating parasite lines are listed in S11 Data. Plaque assays A total of 100 parasites were added to HFF monolayers in a T25 flask and allowed to grow undisturbed for 10 days. Cells were fixed with 100% methanol and stained with crystal violet, then plaques were imaged on a ChemiDoc imaging system (BioRad). Plaque area was measured in FIJI [83]. Differences between strains were determined by one-way analysis of variance (ANOVA) with Tukey’s multiple comparison test. Co-immunoprecipitation mass-spectrometry Immunoprecipitation. HFFs grown to confluency in T175 flasks were pre-stimulated with 2.5 U/ml IFNγ (Bio-Techne, 285-IF-100) for 6 h prior to infection with RHΔKu80 or RHGRA57-HA in triplicate, and 24 h postinfection, infected cells were washed 3× in cold PBS then lysed in cold immunoprecipitation (IP) buffer (10 mM Tris, 150 mM NaCL, 0.5 mM EDTA + 0.4% NP40, pH 7.5 in H 2 O, supplemented with 2× cOmplete Mini EDTA-free Protease Inhibitor Cocktail). Lysates were syringe lysed 6× through a 30 g needle, then centrifuged at 2,000 g for 20 min to remove the insoluble fraction. Soluble fractions were added to 50 μl/sample anti-HA agarose matrix (Thermo), then incubated overnight at 4°C with rotation. The matrix was washed 3 times with IP buffer, then proteins were eluted in 30 μl 3× Sample Loading Buffer (NEB) at room temperature for 10 min. Mass spectrometry. Approximately 20 μl of each IP elution was loaded on a 10% Bis-Tris gel and run into the gel for 1 cm, then stained with InstantBlue Coomassie Protein Stain. Proteins were alkylated in-gel prior to digestion with 100 ng trypsin (modified sequencing grade, Promega) overnight at 37°C. Supernatants were dried in a vacuum centrifuge and resuspended in 0.1% trifluoroacetic acid (TFA), and 1 to 10 μl of acidified protein digest was loaded onto a 20 mm × 75 μm Pepmap C18 trap column (Thermo Scientific) on an Ultimate 3000 nanoRSLC HPLC (Thermo Scientific) prior to elution via a 50 cm × 75 μm EasySpray C18 column into a Lumos Tribrid Orbitrap mass spectrometer (Thermo Scientific). A 70’ gradient of 6% to 40% B was used to elute bound peptides followed by washing and re-equilibration (A = 0.1% formic acid, 5% DMSO; B = 80% ACN, 5% DMSO, 0.1% formic acid). The Orbitrap was operated in “Data Dependent Acquisition” mode followed by MS/MS in “TopS” mode using the vendor supplied “universal method” with default parameters. Raw files were processed to identify tryptic peptides using Maxquant (maxquant.org) and searched against the Toxoplasma (ToxoDB-56_TgondiiGT1_AnnotatedProteins) and Human (Uniprot, UP000005640) reference proteome databases and a common contaminants database. A decoy database of reversed sequences was used to filter false positives, at peptide and protein false detection rates (FDRs) of 1%. T test-based volcano plots of fold changes were generated in Perseus (maxquant.net/perseus) with significantly different changes in protein abundance determined by a permutation-based FDR of 0.05% to address multiple hypothesis testing. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD041352. Protein secondary structure prediction Protein sequences for GRA57, GRA70, and GRA71 from ToxoDB were used to predict boundaries and regions of secondary structure with PSIPRED [84]. Transmembrane domains (TMDs) were assigned using MEMSAT-SVM (within PSIPRED), DeepTMHMM [85], and CCTOP [86], with TMDs only annotated where ≥2 programmes concurred. Regions of predicted coiled-coil were determined using Waggawagga [87], with coiled-coil annotated where the probability score was ≥90. Western blotting Parasites were isolated from host cells by syringe-lysis through 27-gauge needles, passed through 5 μm filters and washed ×1 with cold PBS. Samples were lysed on ice in 1% NP40 IP buffer (10 mM Tris, 150 mM NaCL, 0.5 mM EDTA + 1% NP40, pH 7.5 in H 2 O, supplemented with 1× cOmplete Mini EDTA-free Protease Inhibitor Cocktail) for 30 min, then spun at 2,000 g for 15 min to remove the insoluble fraction. Approximately 10 μg of protein per sample was incubated with 3× loading buffer for 10 min at room temperature, then separated by SDS-PAGE on a NuPAGE 3% to 8%, Tris-Acetate gel. Proteins were transferred to a nitrocellulose membrane using the High Molecular Weight protocol on the Trans-Blot Turbo transfer system (Bio-Rad). Membranes were blocked in 5% milk in 0.05% Tween 20 in PBS for 1 h at room temperature, followed by incubation for 1 h at room temperature with primary antibodies diluted in 1% milk in 0.05% Tween 20 in PBS. Blots were then incubated with appropriate secondary antibodies for 1 h at room temperature. Primary antibodies used were 1:200 mouse anti-T. gondii (Santa Cruz, SC-52255), 1:1,000 rat anti-HA-Peroxidase (Roche, 12013819001), and 1:1,000 rabbit anti-V5 (Abcam, AB_2809347). Secondary antibodies used were 1:10,000 goat anti-mouse HRP (Insight Biotechnologies, 474–1806) and 1:3,000 goat anti-rabbit HRP (Insight Biotechnology, 474–1506). HRP was detected using an enhanced chemiluminescence (ECL) kit (Pierce), visualised on a ChemiDoc imaging system (BioRad). Transmission electron microscopy HFFs were grown to confluency on 13-mm glass coverslips. HFFs were infected with 60,000 parasites per strain for 24 h, then washed 1× with DPBS prior to fixation in 2.5% glutaraldehyde + 4% formaldehyde in 0.1 M phosphate buffer (PB) for 30 min. Samples were washed 2× with 0.1 M PB and stained with 1% (v/v) osmium tetroxide (Taab)/1.5% (v/v) potassium ferricyanide (Sigma) for 1 h at 4°C. Samples were washed 2× in dH2O and were then transferred to a Pelco BioWave Pro+ microwave (Ted Pella, Redding, United States of America) for a further 2 dH2O washes in the Biowave without vacuum (at 250 W for 40 s). The SteadyTemp plate was set to 21°C. In brief, the samples were incubated in 1% (w/v) tannic acid in 0.05 M PB (pH 7.4) (Sigma) for 14 min under vacuum in 2-min cycles alternating with/without 100 W power, followed by 1% sodium sulphate in 0.05 M PB (pH 7.4) (Sigma) for 1 min without vacuum at 100 W. The samples were then washed in dH2O and dehydrated in a graded ethanol series (25%, 50%, 70%, 90%, and 100%, twice each) and in acetone (3 times) at 250 W for 40 s without vacuum. Exchange into Epon 812 resin (Taab) was performed in 25%, 50%, and 75% resin in acetone, at 250 W for 3 min, with vacuum cycling (on/off at 30-s intervals). The samples were transferred to 100% resin overnight before embedding at 60°C for 48 h. A total of 70 nm sections were sliced with a diamond knife on an RMC Powertome Ultramicrotome and sections analysed by a JEM-1400 FLASH transmission electron microscope (Jeol) with Jeol Matataki Flash sCMOS camera. Immunofluorescence assays HFFs were seeded to confluency on 15-mm glass coverslips and infected with 60,000 parasites/well for 24 h. For images shown in S4 Fig, HFFs were pre-stimulated with 100 U/ml IFNγ for 24 h prior to infection. Infected cells were fixed in 4% paraformaldehyde (PFA) for 15 min, then permeabilised in 0.2% Triton-X100 (GRA2 staining) or in 0.1% saponin (GRA3 staining) for 10 min and blocked with 3% bovine serum albumin (BSA) in DPBS for 30 min. Primary and secondary antibodies used for immunofluorescence assays are listed in S13 Data. All antibodies were incubated in 3% BSA in DPBS for 1 h at room temperature, with 3× washes in DPBS between stages. Final secondary staining was combined with DAPI (5 μg/ml). Slides were mounted in ProLong Gold (Thermo Fisher, P36930). Images were acquired on a VisiTech instant SIM (VT-iSIM) microscope using a 150× oil-immersion objective with 1.5 μm z axis steps. Resultant stacks were deconvoluted and processed using Microvolution plugin in FIJI [83]. c-Myc nuclear translocation assays HFFs were grown to confluence in 8-well ibidi μ-slides (Ibidi, 80806). Cells were infected with 30,000 parasites in DMEM with 0.1% FBS. After 24 h, slides were fixed in 4% PFA for 15 min, permeabilised in 0.2% Triton-X100 for 10 min, then blocked in 3% BSA for 30 min. Host c-Myc was stained using 1:800 rabbit anti-cMyc for 2 h at room temperature, followed by 1:1,000 anti-Rabbit-AlexaFluor488 and 5 μg/ml DAPI for 1 h at room temperature. Images were acquired on a Nikon Ti-E inverted widefield fluorescence microscope with a Nikon Plan APO 40×/0.95 objective and Hamamatsu C11440 ORCA Flash 4.0 camera. Host nuclear c-Myc signal of infected cells was measured in FIJI, and the median background c-Myc signal was subtracted for each image. A minimum of 100 infected cells were analysed per strain for each biological replicate. Data is shown as the median fluorescence intensity for each strain relative to RHΔUPRT in each biological replicate. Differences between strains were determined by one-way analysis of variance (ANOVA) with Tukey’s multiple comparison test. Cytation plate reader assays Survival assays. Host cells were seeded in 96-well black imaging plates (Falcon) to confluency, then media was changed to phenol red free DMEM (Gibco, A1443001) with or without 100 U/ml IFNγ (Human:Bio-Techne, 285-IF-100| Mouse: Thermo Fisher, Gibco PMC4031) for 24 h. Parasite strains were syringe lysed and added to host cells at 40,000 parasites/well. Plates were imaged live at 24 h postinfection (MEFs) or 48 h postinfection (HFFs) on a Cytation 5 plate reader using the 4× objective (HFFs) or 20× (MEFs). Total area with signal in the Texas Red channel (Ex/Em 586/647) was measured per well. Differences between strains were determined by paired two-sided t test. Tryptophan supplementation. HFF were seeded as described above, then treated wells were supplemented with 1 mM L-tryptophan (VWR, J62508-09) dissolved in 0.1 N NaOH at the same time as IFNγ stimulation, as described in [44]. Untreated wells had 0.1 N NaOH added as a vehicle control. Data acquisition on a Cytation 5 plate reader was performed as with survival assays. High-content imaging survival assays Host cells were seeded to confluency in 96-well imaging plates (Ibidi). Cells were pre-stimulated for 24 h with 100 U/ml IFNγ according to host species. Cells were infected at an MOI of 0.3 for 24 h, then fixed in 4% PFA and stained with 5 μg/ml DAPI and 5 μg/ml CellMask Deep Red (Invitrogen). Plates were imaged using the Opera Phenix high-content screening system, with 25 images and 5 focal planes acquired per well. Automated analysis of infection phenotypes was performed using Harmony v5 (PerkinElmer) as described in [54]. Data is reported as the mean proportion of each factor (total Toxoplasma number, vacuole size or vacuole number) in IFNγ-treated wells relative to untreated wells. Differences between strains were determined by paired two-sided t test. Vacuole recruitment assays Infections and staining. Host cells were seeded and pre-stimulated as above in 96-well imaging plates (Ibidi). Cells were infected with 80,000 parasites/well and centrifuged at 300 g for 5 min to synchronise infection. At 3 h postinfection, cells were washed 3× in DPBS to remove extracellular parasites, then fixed in 4% PFA for 15 min and blocked with 3% BSA in DPBS for 1 h at room temperature. Extracellular parasites were stained prior to permeabilisation using 1:1,000 rabbit anti-toxo (Abcam, ab138698) and 1:500 goat anti-rabbit-AlexaFluor647 (Life Technologies, A21244). Cells were then permeabilised with 0.2% Triton-X100 for 10 min and re-blocked with 3% BSA. Host marker recruitment was probed for 2 h at room temperature using the following antibodies: mouse anti-total ubiquitin (1:200, Merck, ST1200), rabbit anti-K63 ubiquitin (1:100, Merck, 05–1308), rabbit anti-K48 ubiquitin (1:500, Sigma, ZRB2150), rabbit anti-M1 linear ubiquitin (1:200, Sigma, ZRB2114), or rabbit anti-RNF213 (1:1,000, Sigma, Human Protein Atlas no. HPA003347), followed by donkey anti-mouse-AlexaFluor488 (Thermo, A32766) or donkey anti-rabbit-AlexFluor488 (Thermo, A32790) for 1 h at room temperature. For measurement of K63 recruitment, cells were imaged on a Nikon Ti-E inverted widefield fluorescence microscope with a Nikon Plan APO 40×/0.95 objective, with at least 100 intracellular vacuoles scored/condition. Automated image acquisition and analysis. Plates were imaged using the Opera Phenix high-content screening system, with 25 images and 5 focal planes from −1 to 1 μm with a step size of 0.5 μm acquired per well. Automated analysis was performed using Harmony v5 (PerkinElmer). Recruitment was automatically quantified by first excluding extracellular parasites, then measuring AF488 signal within the vacuole region (1.5 μm boundary of vacuole) and the cytoplasmic region around the vacuole (2 to 3 μm boundary of vacuole). Parasites with AF488 signal higher within this radius relative to the cytoplasmic background of each infected cell were classed as ubiquitin-recruited. Image acquisition parameters and analysis sequences with antibody-specific thresholds are detailed further in S12 Data. For each well, the percentage of ubiquitin-recruited intracellular vacuoles was determined, and then the mean percentage recruitment was calculated across triplicate wells. A median of 1,900 intracellular vacuoles were quantified per strain for each biological replicate, with a minimum number of 200. Differences between strains were determined by paired two-sided t test. Compound 2 inhibition of egress Host cells were seeded and pre-stimulated as above in 96-well imaging plates (Ibidi). Cells were infected with 400,000 parasites/well and centrifuged at 300 g for 5 min to synchronise infection. At 30 min postinfection, cells were washed 3× in DPBS to remove uninvaded parasites, and then 5 μm Compound 2 (gift from Michael Blackman) was added to treated wells. Cells were returned to the incubator for a further 2.5 h, then fixed and stained for total ubiquitin as described above. RNA-Seq RNA sample preparation. HFFs in T25 flasks were serum starved for 24 h with DMEM containing 0.5% FBS. Treated flasks were simultaneously pre-stimulated for 24 h with 100 U/ml IFNγ (Bio-Techne, 285-IF-100). For infections, parasites were isolated by syringe lysis through a 30-gauge needle, passed through 5 μm filters, centrifuged and resuspended in 0.5% FBS DMEM. HFFs were infected in triplicate with 1 million parasites per flask, and 24 h postinfection, samples were collected by washing each flask ×1 in cold PBS, scraping in 2 ml PBS and transferring to an RNAse free tube. Samples were centrifuged at 2,000 rpm for 10 min, and then lysed in 600 μl RLT buffer. Lysates were homogenised using Qiashredders (Qiagen, 79656), then RNA was isolated using an RNeasy Mini Kit (Qiagen, 74104) according to manufacturer’s instructions. mRNA libraries were prepared using the NEBNext Ultra II Directional PolyA mRNA kit (NEB, E7760L) with 100 ng of input, then sequenced on a NovaSeq (Illumina) using paired end 100 bp reads to a minimum depth of 30 million reads per sample. RNA-Seq data analysis. RNA-seq data was quantified using STAR/RSEM [88] from within the nfcore/rnaseq [89] pipeline (version 3.4), against human and Toxoplasma transcriptomes (GRCh38, annotation release-95 from Ensembl and ToxoDB-59_TgondiiGT1 obtained from ToxoDB). Differential analysis was run across strain and IFNγ treatment groups using DESeq2 (1.36.0) [90], correcting for experimental batch effect in the model. Pairwise comparisons were run and an interactions analysis across the 2 experimental factors against uninfected samples and -IFNγ control group in each case. RSEM counts were imported using tximeta (1.14.1) to account for transcript length, and IHW (1.24.0) was used to control from multiple testing in the differential gene selection (<0.05 FDR). Shrunken log fold changes were calculated using type = “ashr”. RNASeq data have been deposited in the Gene Expression Omnibus (GEO) Database under accession number GSE230866.

Acknowledgments We thank all members of the Treeck laboratory as well as Barbara Clough and Eva Frickel for critical discussions and Stephanie Nofal for critically reading the manuscript. We thank Michael Howell, Rachael Instrell, and Becky Saunders (High-Throughput Screening Science Technology Platform, The Francis Crick Institute, London, United Kingdom) for assistance with sgRNA library preparation. We thank members of the Advanced Sequencing, High Throughput Screening, Electron Microscopy, Proteomics and Cell Services Science Technology Platforms at the Francis Crick Institute for support. We thank Bishara Marzook for assistance with the Cytation 5 plate imager, Jean Francois Dubremetz for providing the GRA3 antibody, Felix Randow and Ana Crespillo-Casado for providing the MEFs, and Matthew Cottee for assistance with protein structural analysis. We acknowledge ToxoDB (http://toxodb.org/) for providing an invaluable resource that made this work possible.

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