TgTEP localizes in the cytoplasm and is essential for parasite survival. (A) Scheme of strategy to determine the localization of TgTEP. A GFP-nanobody fused to a Halo-tag was stably expressed within the parasites by replacing the UPRT locus. This nanobody has no target sequence and therefore localizes within the cytoplasm. In cases where no GFP, YFP, or SYFP2 are accessible within the cytoplasm, no colocalization with the nanobody occurs, and the nanobody signal remains diffuse within the cytoplasm (left panel). In cases where GFP, YFP, or SYFP2 are present within the cytoplasm, the nanobody binds to the fluorescent tag, resulting in colocalization (right panel). (B and C)TgTEP-SYFP2 colocalizes with the cytosolic nanobody. (D–G) FRM-2-SYFP2 (positive control), SAG1-YFP, and TgTEP-mCherry (negative controls) confirm selective binding of the GFP-nanobody. (H) BFA disrupts Golgi, redistributing both TgTEP and SortLR in all cases (100%). All scale bars are 5 μm. (I) 7-day plaque assays were done with LoxP-TgTEP parasites in the presence of 50 nM rapamycin or vehicle control (DMSO). Knockout (KO) mutants (+rapamycin) did not form plaques in the host cell monolayer. Scale bars are 1.5 mm. (J) Replication assays were performed after the induction of parasites with 50 nM rapamycin or DMSO for a period of 48 h. The number of knockout parasites per vacuole was significantly lower than that of WT parasites. (K) Egress assays were done in the presence of 50 nM rapamycin or DMSO (−/+ Rapa). Egress was either allowed to occur naturally or was induced using calcium ionophore A23187 (−/+ CI). The percentage of successfully egressed parasites was significantly lower for the knockout mutants. All assays were done thrice, with a minimum of 100 parasites/vacuoles quantified per condition per replicate. All data are plotted as mean ± SD. One-way ANOVA with Tukey’s multiple comparison test were performed. Color-coded P values in J represent the vacuoles compared. P values are represented as follows: ns ≥ 0.05; * = 0.01 - 0.05; ** = 0.001 – 0.01; *** = 0.0001 – 0.001; **** ≤0.0001.
Figure 3.

TgTEP localizes in the cytoplasm and is essential for parasite survival. (A) Scheme of strategy to determine the localization of TgTEP. A GFP-nanobody fused to a Halo-tag was stably expressed within the parasites by replacing the UPRT locus. This nanobody has no target sequence and therefore localizes within the cytoplasm. In cases where no GFP, YFP, or SYFP2 are accessible within the cytoplasm, no colocalization with the nanobody occurs, and the nanobody signal remains diffuse within the cytoplasm (left panel). In cases where GFP, YFP, or SYFP2 are present within the cytoplasm, the nanobody binds to the fluorescent tag, resulting in colocalization (right panel). (B and C)TgTEP-SYFP2 colocalizes with the cytosolic nanobody. (D–G) FRM-2-SYFP2 (positive control), SAG1-YFP, and TgTEP-mCherry (negative controls) confirm selective binding of the GFP-nanobody. (H) BFA disrupts Golgi, redistributing both TgTEP and SortLR in all cases (100%). All scale bars are 5 μm. (I) 7-day plaque assays were done with LoxP-TgTEP parasites in the presence of 50 nM rapamycin or vehicle control (DMSO). Knockout (KO) mutants (+rapamycin) did not form plaques in the host cell monolayer. Scale bars are 1.5 mm. (J) Replication assays were performed after the induction of parasites with 50 nM rapamycin or DMSO for a period of 48 h. The number of knockout parasites per vacuole was significantly lower than that of WT parasites. (K) Egress assays were done in the presence of 50 nM rapamycin or DMSO (−/+ Rapa). Egress was either allowed to occur naturally or was induced using calcium ionophore A23187 (−/+ CI). The percentage of successfully egressed parasites was significantly lower for the knockout mutants. All assays were done thrice, with a minimum of 100 parasites/vacuoles quantified per condition per replicate. All data are plotted as mean ± SD. One-way ANOVA with Tukey’s multiple comparison test were performed. Color-coded P values in J represent the vacuoles compared. P values are represented as follows: ns ≥ 0.05; * = 0.01 - 0.05; ** = 0.001 – 0.01; *** = 0.0001 – 0.001; **** ≤0.0001.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal