MYR1 and MYR1-dependent factors are not contributing to in vitro Toxoplasma survival in IFN-γ-activated macrophages.

(a) BMDMs were stimulated with 100 U/ml IFN-γ for 24 h or left untreated, before infection with mCherry-expressing Toxoplasma strains for 24 h. Cells were fixed and imaged by high-content imaging, and the percentage of infected cells in IFN-γ-treated BMDMs compared to untreated controls is shown. WT refers to PruΔUPRT. The box-plot shows the median value and the whiskers show minimum and maximum values. Significance was tested with the One-way Anova test with the Benjamini, Krieger and Yekutieli FDR correction, n=5. (b) Schematic of the flow cytometer-based growth competition assay of an IFN-γ-dependent effector (e.g. GRA12). HFFs and BMDMs were co-infected with equal amounts of colourless WT and mCherry-expressing ΔGRA12 or ΔMYR1 strains. BMDMs were stimulated with 100 U/ml IFN-γ for 24 h or left untreated before infection. The mCherry signal was quantified by flow cytometry analysis and the ratio between the strains after two passages (output) was compared to the input. (c) Growth of competing mutant and WT parasites was assessed by flow cytometry. The normalised ratios of output versus input are shown. The average of two independent experiments with technical triplicates is shown. (d) Violin plot of the plaque size of parental PruΔKU80 and derived PruΔMYR1 strains. The bar represents the median value and representative images are reported on the right. Significance was tested using a two-tailed unpaired Welch’s t-test, ** p<0.01, **** p<0.0001, n=2.

ΔMYR1 parasites expand in the murine host and form cysts in vivo.

(a) Mice were infected i.p. with luciferase-expressing Toxoplasma strains, WT-Luc or ΔMYR1-Luc, and whole-body intravital imaging was performed at days 3, 5 and 7 p.i. (b) Representative whole-body intravital imaging. (c) Graph shows the total bioluminescence signal converted to a logarithmic scale from two independent experiments. Statistical differences between the two strains at each timepoint were tested with the Mixed-effects model (REML) with Sidak post-hoc tests (continued lines). Significance for the ΔMYR1-Luc growth over time was tested with a One-Way Anova test (dotted line). Number of mice per group: day 3 p.i.: n=14; day 5 p.i.: n=11; day 7 p.i.: n=8. * p<0.05, *** p<0.001. (d) Representative fluorescent image of a brain cyst from a mouse infected with ΔMYR1-Luc. mCherry-expressing ΔMYR1-Luc parasites were detected by microscopy. Cyst wall was stained with the FITC-conjugated lectin DBA. The scale bar represents 25 µm.

The in vivo growth defect of ΔMYR1 is rescued by the presence of MYR1-competent parasites, independently of functional host adaptive immunity.

(a) Mice were infected i.p. with a mixed inoculum of Toxoplasma tachyzoites, containing a 20:80 ratio of luciferase-expressing ΔMYR1-Luc to WT or ΔMYR1 strains that do not express luciferase. Growth of ΔMYR1-Luc was monitored at 3, 5 and 7 days p.i. by whole-body intravital imaging. (b) Representative whole-body intravital imaging. (c) Total bioluminescence signal converted to a logarithmic scale from mice co-infected with ΔMYR1-Luc:WT (n=18) or ΔMYR1-Luc:ΔMYR1 (n=17). Graph shows cumulative data from four independent experiments. Significance was tested with a Two-way Repeated Measures ANOVA with Sidak post-hoc tests. (d) IL-12p40, CCL2/MCP-1, TNF and IFN-γ levels were detected in peritoneal lavage and serum of animals infected with mixed Toxoplasma inoculum (20:80 ratio) at day 7 p.i. by ELISA. The violin-plots show the median (continued black line) and quartiles (dotted grey lines). Significance was tested with the Welch’s t-test (TNF, CCL2/MCP-1 and IL-12p40 ELISAs) and Mann-Whitney test (IFN-γ ELISAs). Samples from 2 to 3 independent experiments were assayed. Number of samples for CCL2: WT n=13 and ΔMYR1 n=12. Number of samples for other cytokines: peritoneal lavage samples: WT n=8 and ΔMYR1 n=7; serum samples: WT n=4 and ΔMYR1 n=3. Data with higher differences between inocula (CCL2 and IFN-γ) are displayed in a logarithmic scale in SI Appendix Fig. S1d-f. (e) Representative whole-body intravital imaging at day 7 p.i. of C57BL/6Ntac (WT) or RAG2-deficient (Rag2-/-) mice infected i.p. with a mixed inoculum containing a 20:80 ratio of luciferase-expressing ΔMYR1-Luc tachyzoites to WT or ΔMYR1 strains that do not express luciferase. (f) Total bioluminescence signal converted to a logarithmic scale from mice co-infected with ΔMYR1-Luc:WT (n=10) or ΔMYR1-Luc:ΔMYR1 (n=8). Graph shows cumulative data from two independent experiments. Significance was tested with the Multiple Mann-Whitney test with a False Discovery Rate approach by a Two-stage step-method. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.

Model illustrating paracrine rescue of ΔMYR1 parasites in pooled CRISPR screens.

Left panel: In single-strain infections ΔMYR1 parasites grow slowly within the peritoneum and are cleared. Right panel: Growth of ΔMYR1 parasites within a CRISPR pool is rescued by the presence of MYR-competent parasites. In cells infected with MYR1 competent parasites, MYR-dependent effectors enter the host cell, leading to secretion of cytokines that promote a favourable immune environment. MYR1 mutants are able to proliferate under these conditions (i.e are rescued in trans). However, Toxoplasma virulence factor mutants required for survival within a cell (cell-autonomous), such as GRA12, can not be rescued in trans.

(a) List of primers used in the study. (b) PCR validation of the PruΔIST strain compared to parental PruΔKu80 (WT). (c) PCR validation of the luciferase-expressing WT-Luc and ΔMYR-Luc strains compared to their respective parental strains PruΔKu80 (WT) and PruΔMYR1 (ΔMYR1). (d, e and f) IFN-γ at peritoneal lavage (d) and CCL2 at both peritoneal lavage (e) and serum (f), were detected by ELISA at day 7 p.i. with mixed Toxoplasma inocula (20:80 ratio) of luciferase-expressing ΔMYR1-Luc to WT or ΔMYR1 strains that do not express luciferase. The violin-plots display data shown in Fig. 3d converted to a logarithmic scale, with the median (continued black line) and quartile values (dotted grey lines) highlighted. Significance was tested with the Mann-Whitney test for IFN-γ, and by Welch’s t-test for CCL2 data. The number of biological replicates (n) in each graph is the same as mentioned in the original graphs on Fig. 3d. *** p<0.001, **** p<0.0001.