Fig 1.
Ingestion of host cytosolic proteins is not upregulated in Δgra17 parasites.
CHO cells were transfected with a GFP-expressing plasmid and 24 h after transfection cells were infected with indicated parasites treated or not with 1 μM LHVS. 24 h p.i., parasites were harvested and the percentage of parasites containing GFP in the vacuolar compartment (VAC) was determined with a fluorescent microscope. Shown are means±SD from three independent experiments. Significance was assessed by one way ANOVA with Tukey’s multiple comparison test. * Indicates P<0.05. ns indicates “not significant”.
Fig 2.
Genome-wide CRISPR screen identifies Toxoplasma genes that are synthetically sick/lethal with GRA17.
A) CRISPR synthetic lethality screen procedure. Wild-type (WT) or Δgra17 parasites expressing Cas9 were transfected with CRISPR plasmids containing 10 different sgRNAs against each of 8,156 Toxoplasma genes. The pool of parasite mutants was passaged in HFFs for four rounds under pyrimethamine selection to select for parasites that integrated the sgRNA-containing plasmid. The abundance of sgRNAs at the 3rd and 4th passage was determined by Illumina sequencing and used to calculate fitness scores to identify genes that have a fitness defect in Δgra17 but not in WT parasites. B) Identification of genes with a larger fitness defect when deleted in Δgra17 vs. wild-type parasites. WT and Δgra17 Cas9-expressing parasites were electroporated with two plasmids containing a pyrimethamine resistance cassette and a different sgRNA targeting one of the hits from the synthetic lethality screen (Table 1). After electroporation 5,000 parasites were added to 6-well plates containing HFFs and 3 μg/ml (1μM) pyrimethamine and incubated for 8 days after which the number of plaques were counted. Plotted are the percentage of plaques formed in WT or Δgra17 parasites after transfection with two sgRNAs targeting the indicated genes relative to the number of plaques after transfection with the SAG1-targeting sgRNAs. Error bars are SD (n = 3 biological replicates). ND = not detected. C-D) GRA45 and MYR4 are synthetically sick with GRA17. WT or knockout parasite strains were used to infect HFFs and five days post infection the plaque areas were quantified. Relative parasite growth of knockout parasites was calculated relative to the plaque area of WT parasites (set at 100%). The results are shown as mean ± SD from three independent experiments. Statistical analysis was done with One-way ANOVA followed by Tukey’s multiple comparison test (*p < .05, **p < .01).
Table 1.
Hits from the CRISPR screen that were selected for confirmation.
Phenotype scores for the indicated parasite genes in Δgra17 vs. WT parasites at the 4th passage are shown. Also included are the P-values (average from 3rd/4th passage), which were calculated using the MAGeCK algorithm. For all data and further details, please refer to S1 Table.
Fig 3.
Localization of GRA17 and GRA23 in knockout parasites.
HFFs were infected with the indicated parasite strains transiently expressing GRA17-HA, GRA17-V5, or GRA23-HA at an MOI of 0.5 for 24 h. Representative images of A) GRA17 and B) GRA23 localization in the different strains. Scale bar = 8μm. The percentage of vacuoles with PV lumen, PVM, or partial PVM staining of C) GRA17 or D) GRA23 was quantified. Statistical significance was determined by Two way-ANOVA with Dunnett’s multiple comparisons test. (****p < .0001, n = 12 for WT, n = 3 for other strains).
Fig 4.
Localization of GRA5 and GRA7 in knockout parasites.
HFFs were infected with the indicated parasite strains at an MOI of 0.5 for 24 h. Representative images of A) GRA5 and B) GRA7 localization in the different strains. Scale bar = 9μm. The percentage of vacuoles with PV lumen, PVM, or partial PVM staining for C) GRA5 or D) GRA7 was quantified. Statistical significance was determined by Two way-ANOVA with Dunnett’s multiple comparisons test (****p < .0001, n = 3).
Fig 5.
Δgra72 parasites form bubble vacuoles with decreased PVM permeability to small molecules.
A) HFFs were infected with indicated parasite strains at an MOI of 1 for 36 h (WT = wild-type, Comp = Δgra72+GRA72). Shown are representative examples of a bubble (abnormal vacuole) in Δgra72 parasites and normal vacuole in WT and complemented parasites. B) Vacuoles were quantified as normal or abnormal from at least 20 microscopic fields. Vacuoles with abnormal morphology include bubble vacuoles, enlarged collapsed vacuoles and vacuoles containing opaque tachyzoites. Both normal and abnormal vacuoles were quantified from three different passages (infections). Two way-ANOVA with Dunnett’s multiple comparison test was used to determine statistical significance (****p < .0001, n = 3) C) HFFs were infected with indicated parasite strains for 24 h and subsequently pulsed with CDCFDA for 10 minutes after which the dye was washed away and vacuoles were imaged. Shown are representative images from the wild-type and complemented strain showing normal permeability to CDCFDA and indicated knockout parasite strains with reduced permeability to CDCFDA. D) The percentage of CDCFDA-fluorescent vacuoles was quantified for each strain. At least 50 vacuoles per well were quantified and identified as CDCFDA-positive or negative. Data are displayed as average (±SD) values from 3 independent experiments. One-way ANOVA with Tukey’s multiple comparison test was used to determine significance (****p < .0001, n = 3). E) HFFs were infected with indicated parasite strains (MOI of 1) for 24 h. The parasitophorous vacuole was stained with anti-GRA5 antibody and parasites were stained with anti-SAG1 antibody and the number of parasites/vacuole was determined for ~100–200 vacuoles per experiment. Data are displayed as average (±SD) values from 3 independent experiments. A two-way ANOVA with Dunnett’s multiple comparison test was used to determine significance.
Fig 6.
AlphaFold-multimer predicts GRA17 and GRA72 as pore like assemblies.
A) Cartoon depiction of Pf-EXP2 structure (aa 27–235 are modelled) extracted from the full PTEX cryo-EM structure in the extended state (PDB id: 6E10). On the left, the pore forming complex is seen from the bottom and monomers are colored individually. On the right, the Pf-EXP2 complex is flipped by 90°C and depicted in a hybrid manner, with both a cartoon view with N and C-terminal ends indicated and by a lipophilicity surface representation. An under view of the pore channel is also displayed with corresponding dimensions. B) AlphaFold-multimer prediction of a heptameric assembly of GRA17. On the left, the full length GRA17 sequence was used for the prediction while starting on the middle and on the right, a minimized structural core (aa 113–300) was used for the prediction. Cartoon depictions are colored (from purple to green) using the predicted local distance difference test (pLDDT) produced by AlphaFold. The minimized structure on the right is also flipped by 90°C and half displayed using the surface lipophilicity representation as in panel A. N and C-terminal ends are indicated on the cartoon half while an under view of the pore channel is also displayed with corresponding dimensions. C) AlphaFold-multimer prediction of a hexameric assembly of GRA72 (aa 49 to 356 and aa 148 to 248) following the same representations codes as in B. All representations were produced using ChimeraX.
Fig 7.
Δgra72 parasites have reduced in vivo virulence.
A. CD1 mice were infected intraperitoneally with 5,000 tachyzoites of WT, Δgra72 or Δgra72 +GRA72-HA parasites (all in the ME49 type II strain). Mice were monitored and weighed regularly throughout the infection period and the weight is plotted as the average change in body weight for each cohort, where the weight on the day before infection was considered as 100%. The statistical significance was performed by one-way ANOVA with Tukey’s multiple comparison test from time points from day 0—day 20 (* P< 0.05, ** P<0. 01, *** P< 0.001). B. The post-infection survival of mice was monitored for 30 days. The data are displayed as Kaplan–Meier survival curves (data presented for wild-type or Δgra72 parasites are pooled from two independent experiments, each involving n = 5 mice, for a cumulative total of n = 10 mice, and one experiment with n = 5 for mice infected with Δgra72 +GRA72-HA parasites). The statistical significance was determined by the Log-rank (Mantel–Cox) test.