Figure 1.
(A) Plasmid map of transfer vectors pDMG and pDMG-TgROP18, respectively. DHFR, dihydrofolate reductase-thymidylate synthase; MCS, multiple cloning site; GFP, green fluorescent protein. (B) The recombinant N. caninum stably expressing TgROP18 was isolated by flow cytometry. (C) IFA Localization of TgROP18 in Nc1-TgROP18. The recombinant N. caninum stably expressing TgROP18 was confirmed by IFA using mouse anti-rTgROP18 serum as the primary antibody. Scale bar, 5 µm.
Figure 2.
Identification of Nc1-TgROP18.
(A) Electrophoresis of the PCR products. Both the GFP and TgROP18 target genes (717 bp and 1,662 bp, respectively) were amplified from the genomic DNA of the Nc1-TgROP18 strain. (B) qRT-PCR comparison of TgROP18 expression in RH strain and Nc1-TgROP18 strain. Data are mean ± SD (error bars) of three independent experiments. (C) and (D) Expression of TgROP18 in the cloned parasites was confirmed by Western blot analysis. The TgROP18 and GFP fusion protein (∼87 kDa) was recognized by both anti-GFP and anti-TgROP18 polyclonal antibodies.
Figure 3.
The indicated strains grew on HFF cells for 7 days before fixation and staining with Crystal violet. The Nc1 strain incubated for 30°C was used as the negative control. Three independent experiments were performed and results of one representative experiment are shown here. Scale bar, 200 µm.
Figure 4.
Phenotype assays of Nc1-TgROP18.
(A) Transmigration assay in the Transwell system. Data are represented as mean ± SD (error bars) of three independent experiments. (B) Gliding motility assay. The trails were stained with the anti-TgSAG1 antibody (RH strain) or anti-NcSRS2 antibody (Nc1, Nc1-GFP and Nc1-TgROP18 strains). The arrow indicates a trail. Scale bar, 5 µm. (C) Cell invasion assay showed no difference in cell invasion between the transgenic Nc1-TgROP18 strain and the untransfected Nc1 or T. gondii RH strain. Data are mean ± SD (error bars) of three independent experiments. (D) Intracellular replication assay of the transgenic Nc1-TgROP18 compared to the Nc1 strain, Nc1-GFP strain and the RH strain. Asterisks indicate statistically significant results (*P≤0.005; **P≤0.001), as determined with the Student's t test. Data are mean ± SD (error bars) of three independent experiments.
Figure 5.
Mice (n = 5) were injected with 10, 103 or 106 tachyzoites and were monitored for 30 days. All mice injected with the Nc1-TgROP18 strain and T. gondii RH strain died within 16 days post infection, compared with no death of mice infected with Nc1 wide-type or Nc1-GFP tachyzoites. Three independent experiments were performed and one representative is shown here.
Figure 6.
(A) and (B) Localization of phosphorylated Irga6 in IFNγ-stimulated Ana-1 cells infected with Nc1 wide-type strain or transgenic Nc1-TgROP18 strain, respectively. TgROP18 localized with mouse Anti-TgROP18 serum (FITC, green), Irga6 localized with rabbit Anti-Irga6 phosphopeptide Ab T102-555 (Texas Red, red), and nuclei stained with DAPI (blue). Scale bar, 2.5 µm.