FIG 4.

Effect of TLN4-deficient parasites on the lytic cycle. (A) Plaque assays show smaller plaques in Δtln4 parasites. One hundred parasites of each strain were inoculated into 6-well plates and allowed to grow for 7 days undisturbed. Wells were stained with crystal violet. (B) Individual plaque sizes were measured using ImageJ. Data represent three independent biological experiments with triplicate samples within each experiment. A minimum of 500 plaques were counted per strain. Error bars represent standard errors of the means (SEM), and statistics were performed using an unpaired, two-tailed, Student's t test. (C) Parasites lacking TLN4 show normal invasion. HFF cells in 8-well chamber slides were inoculated with parental, Δtln4, or Δtln4TLN4 parasites and allowed to invade for 20 min prior to fixation. Wells were differentially stained with SAG1 antibodies to detect attached or invaded parasites per host cell nucleus. A minimum of 250 host cells were counted per strain. Data in the invasion (C) and replication (D) graphs represent means ± SEM from three independent experiments, each with triplicate samples. At least 8 fields of view were counted per well. (D) Parasites lacking TLN4 replicate normally. Parasites were inoculated into 8-well chamber slides and allowed to replicate for 17 h or 26 h prior to fixation and enumeration of parasites per vacuole. A minimum of 250 vacuoles were counted per strain per time point. (E) Δtln4 parasites show normal modes of gliding motility. Video microscopy to enumerate the types of gliding motility shows the percentages of immobile parasites and parasites performing twirling, circular, and helical gliding in all strains tested. Graphs indicate the means ± SEM from three independent experiments. *, P ≤ 0.05 by Student's t test. (F) TLN4 does not play a role in acute virulence. Swiss-Webster mice were infected intraperitoneally with 10 or 100 tachyzoites of WT or Δtln4 parasites, and survival time was enumerated; 12 mice were infected for each strain and inoculum.