Figure 6. Mutagenesis of TgPKG recapitulates the phenotype of the TgATPaseP-GC mutant.
(A–F) Phenotyping of the TgPKG mutant in comparison with its progenitor and parental strains. For making of the mutant, refer to Fig S7. (A) Images demonstrating the expression of TgPKG-HA3’IT in the progenitor strain, and its down-regulation in the mutant. The parental strain served as a negative control. Intracellular parasites (24 h postinfection) were stained with α-HA and α-TgGap45 antibodies. The merged image includes DAPI-stained host and parasite nuclei in blue. Scale bars represent 2 µm. (B) Immunoblot depicting the expression of TgPKG isoforms in clonal mutants (C1 and C2) along with the progenitor and parental strains. Extracellular tachyzoites (2 × 107) of each strain were subjected to protein isolation followed by immunoblotting with α-HA antibody. Expression of 112 and 135-kD isoforms in the progenitor and mutants, but not in the parental strain, confirms efficient 3′-HA tagging and successful knockdown of the protein, respectively. TgRop2 served as loading control. (C) Plaque assay revealing comparative growth of the mutant, progenitor, and parental strains. The plaque area is shown in arbitrary units (a. u.). A total of 140–170 plaques of each strain were scored from three assays. (D) Replication rates of indicated strains during early (24 h) and late (40 h) cultures. Tachyzoites proliferating in their vacuoles were stained with α-TgGap45 antibody and counted from 400 to 500 vacuoles for each strain (n = 3 assays). (E, F) Invasion and egress of the designated parasites as judged by dual-color staining. Intracellular tachyzoites were immunostained red using α-TgGap45 antibody, whereas extracellular ones appeared two-colored (red and green), stained with both α-TgGap45 and α-TgSag1 antibodies. In total, 1,000–1,200 parasites were evaluated to score the invasion rate of each strain (n = 5 assays). The percentage of ruptured vacuoles at indicated periods was determined by observing 400–500 vacuoles of each strain from three experiments. (G) Motile fraction and trail lengths of the indicated parasite strains. Extracellular parasites immunostained with α-TgSag1 were analyzed for the motile fraction (600 parasites) and trail lengths (100 parasites) (n = 3 assays). *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; and ****P ≤ 0.0001.