Table 1.

Toxoplasma genes that determine fitness in naïve or IFNγ-activated macrophages.

Genes with a fitness defect in naïve macrophages
Gene ID	Product description	Naïve BMDM vs. HFF Control fitness (mean ± SEM)	p value (one-sided Wilcoxon signed-rank test)	Cohen’s d	IFNγ vs. Naïve BMDM fitness	In vitro phenotype in HFFs53	In vivo fitness55,84
TGGT1_249580	TgApiAT6-3	−4.2 ± 1.1	0.001	7.6	−0.2	0.5	N/A
TGGT1_247360	PAP2 superfamily protein	−3.2 ± 1.0	0.002	5.8	−1.2	1.6	N/A
TGGT1_250880	Kinase, pfkB family protein (Adenosine Kinase)	−1.8 ± 0.4	0.002	3.4	1.2	1.4	N/A
TGGT1_290860	TgApiAT6-2	−1.5 ± 1.2	0.03	2.8	0.5	1.0	N/A
TGGT1_248560	Hypothetical protein; homology to 26 S Proteasome regulatory subunit RPN2 (Swiss-Model)	−1.5 ± 0.5	0.003	2.8	0.1	0.3	N/A
TGGT1_308075	Hypothetical protein; homology to DNAse Pyocin AP41 (Swiss-Model)	−1.3 ± 0.2	0.003	2.5	0.7	1.5	N/A
TGGT1_224840	3'5’-cyclic nucleotide phosphodiesterase domain-containing protein	−1.3 ± 0.3	0.004	2.4	0.3	0.4	N/A
TGGT1_262650	WD domain, G-beta repeat-containing protein	−1.1 ± 0.4	0.007	2.1	0.2	0.4	N/A
TGGT1_269390	CRAL/TRIO domain-containing protein	−1.0 ± 0.6	0.03	2.0	−0.1	0.1	N/A

Genes with a fitness defect in IFNγ-activated macrophages
Gene ID	Product description	IFNγ vs. naïve BMDM fitness (mean ± SEM)	p value (one-sided Wilcoxon signed-rank test)	Cohen’s d	Naïve BMDM vs. HFF control Fitness	In vitro phenotype in HFFs53	In vivo fitness55,84
TGGT1_269620	DNA repair protein-like (Swiss-model)	−4.0 ± 1.6	0.004	2.9	1.5	−0.3	N/A
TGGT1_209500	DNA repair protein-like (HHpred)	−3.6 ± 1.8	0.006	2.7	0.4	0.1	N/A
TGGT1_295340	UV excision repair protein Rad23 protein	−3.5 ± 1.0	0.003	2.6	1.4	−0.4	N/A
TGGT1_316250	GRA45	−3.3 ± 0.4	0.002	2.4	1.0	1.2	N/A
TGGT1_232670	Cytohesin-3-like; GEF of Arf6 (Swiss-Model)	−3.3 ± 0.8	0.003	2.4	0.3	1.5	N/A
TGGT1_321530	Cathepsin L (CPL)	−3.3 ± 0.9	0.003	2.4	1.7	0.7	N/A
TGGT1_263560	Putative GRA (Supplementary Fig. 5i)	−3.0 ± 1.2	0.006	2.2	1.5	0.5	N/A
TGGT1_208370	GRA46	−3.0 ± 1.6	0.008	2.1	1.5	1.8	N/A
TGGT1_228300	CCDC25 protein	−2.9 ± 0.6	0.003	2.1	0.3	−1.1	N/A
TGGT1_215220	GRA22	−2.9 ± 1.8	0.02	2.1	1.1	1.0	Peritoneum
TGGT1_314500	SUB2	−2.8 ± 1.4	0.01	2.0	0.8	0.3	N/A
TGGT1_227560	Putative IWS1 transcription factor	−2.7 ± 1.7	0.02	1.8	1.3	−0.7	N/A
TGGT1_200290	ROM1	−2.4 ± 1.3	0.01	1.7	0.2	0.8	N/A
TGGT1_240980	Cytoplasmic dynein-like (Swiss-Model/HHpred)	−2.4 ± 0.9	0.007	1.7	0.3	−1.0	N/A
TGGT1_276940	Putative ribosome associated membrane protein RAMP4	−2.4 ± 1.4	0.02	1.7	−0.4	0.4	N/A
TGGT1_222050	Putative methylcitrate synthase (Swiss-Model)	−2.2 ± 1.3	0.03	1.5	0.6	0.7	N/A
TGGT1_269950a	Putative GRA (ToxoDB.org); homology to QSOX (HHpred)	−2.1 ± 1.2	0.2	1.4	1.4	0.4	Peritoneum

Upper panel: nine top hits, selected from 193 naïve BMDM fitness-conferring genes, with at least four sgRNAs present in the last passage of HFF control and ≥2-fold lower fitness (fitness score ≤ −1) in naïve BMDMs vs. HFF control. Genes with a mean Naïve BMDM fitness > −1.5 and a mean HFF control fitness < −1.5 were removed.

Lower panel: 16 top hits (not including TGGT1_269950), selected from 232 IFNγ fitness-conferring genes, with at least four sgRNAs present in the 3rd passage of naïve BMDMs and a ≥4-fold lower fitness (fitness score ≤ −2) in IFNγ vs. naïve BMDMs. Genes with a mean IFNγ fitness > −0.5 and a mean naïve BMDM fitness < 0 were removed. The genes are ranked according to the mean fitness (naïve BMDM vs. HFF control, upper panel; IFNγ vs. naïve BMDM, lower panel). See Supplementary Data 1 for the complete list.

^aTGGT1_269950 was confirmed as an IFNγ fitness-conferring gene (Fig. 2f), although it does not fit the above criteria.