Primer sequences used in this work.

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.</div

Opera Phenix image acquisition parameters and Harmony analysis sequence used for automated ubiquitin recruitment analysis.

Opera Phenix image acquisition parameters and Harmony analysis sequence used for automated ubiquitin recruitment analysis.</p

RNASeq of infected HFFs.

Differential gene analysis and interaction analysis of human-aligned transcripts. IFNγ treatment is denoted as yes or no. For the interaction analysis, sheet names represent the IFNγ effect for the first strain relative to the IFNγ effect for the second strain (i.e., WT|IFN_vs_dGRA57|IFN represents +IFNγ vs -IFNγ in the RHΔKu80 group relative to +IFNγ vs -IFNγ in the RHΔGRA57 group). (XLSX)</p

GRA57 is an intravacuolar protein that contributes to parasite survival of IFNγ in HFFs.

(A) IFA of RHΔGRA57 and RHΔGRA57::GRA57-HA lines generated for this study. HA-tagged GRA57 co-localises with GRA2—a marker of the IVN. Scale bar = 3 μm. (B) Western blot analysis of GRA57-HA shows it is a 250 kDa protein that is relatively highly expressed. (C and D) Live restriction assays in (C) HFFs and (D) MEFs using mCherry fluorescence area as a readout for parasite survival. Host cells were pre-stimulated for 24 h with 100 U/ml IFNγ or left untreated then infected in technical triplicate with the indicated parasite strains for 48 h (HFFs) or 24 h (MEFs) at an MOI of 0.3. Infected cells were then imaged live on a Cytation plate reader. Total mCherry signal area per well was measured to determine parasite growth in IFNγ-stimulated relative to unstimulated cells. Data displayed as median survival with individual biological replicates overlayed. p-Values were calculated by paired two-sided t test. **, p S3 Data. HFF, human foreskin fibroblast; MEF, mouse embryonic fibroblast.</p

GRA57-HA partially co-localises with the PVM marker GRA3.

GRA57-HA partially co-localises with the PVM marker GRA3.</p

Deletion of GRA57, GRA70, or GRA71 leads to reduced host ubiquitin recruitment to the PVM.

(A) Schematic of automated high-content imaging analysis pipeline to determine ubiquitin recruitment levels. (B, C) Recruitment of total host ubiquitin (FK2) to Toxoplasma vacuoles in (B) HFFs and (C) MEFs. Host cells were pre-stimulated with 100 U/ml IFNγ for 24 h, infected with indicated lines for 3 h prior to fixation and staining for total ubiquitin. Recruitment of ubiquitin was automatically counted using high-content imaging and analysis. (D) Recruitment of total ubiquitin (FK2), K63-linked ubiquitin, linear ubiquitin (M1), and the E3 ligase RNF213 to Toxoplasma vacuoles. HFFs were pre-stimulated with 100 U/ml IFNγ for 24 h, infected with indicated lines for 3 h prior to fixation and staining. Recruitment was automatically quantified for FK2, M1, and RNF213. K63 recruitment was manually scored, with minimum 100 vacuoles scored per condition. p-Values were calculated by paired two-sided t test. *, p p p p S9 Data. HFF, human foreskin fibroblast; MEF, mouse embryonic fibroblast; PVM, parasitophorous vacuole membrane.</p

Raw data for Compound 2 egress inhibition assays.

(A) Percentage of vacuoles with ubiquitin recruitment +/− IFNγ and +/− Compound 2 per biological replicate. (B) Survival percentages at 3 h postinfection calculated from number of intracellular Toxoplasma per biological replicate. (XLSX)</p

GRA57 deletion does not affect formation of the IVN.

HFFs monolayers were infected with the indicated strains for 24 h prior to fixation and preparation for transmission electron microscopy (TEM). (TIF)</p

Antibodies used for immunofluoresence assays.

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.</div

GRA57/GRA70/GRA71 all contribute equally to resisting IFNγ-induced vacuole destruction in human fibroblasts.

(A–C) HFFs cells were pre-stimulated for 24 h with 100 U/ml IFNγ or left untreated then infected in technical triplicate with the indicated parasite strains for 24 h at an MOI of 0.3. Parasite survival was quantified through automated high-content imaging. (A) Parasite survival calculated as total number of Toxoplasma in IFNγ-stimulated cells as a percentage of the total in unstimulated cells. (B) Parasite survival calculated as number of vacuoles in IFNγ-stimulated cells as a percentage of the number in unstimulated cells. (C) Parasite survival calculated as mean vacuole area per well [px2] in IFNγ-stimulated cells as a percentage of the area in unstimulated cells. Data displayed as median survival with individual biological replicates overlayed. p-Values were calculated by paired two-sided t test. *, p p S5 Data. HFF, human foreskin fibroblast.</p