Extended Data Fig. 3: Depletion of Nd6 or Nd9 in T. gondii parasites does not affect replication, egress, gliding motility nor attachment.
From: An Alveolata secretory machinery adapted to parasite host cell invasion
a, Plaque assay. Left: ΔKu80-Tir1 (Ctrl) ± IAA vs Tgnd6 iKD ± IAA. Right: ΔKu80-TaTi (Ctrl) ± ATc vs Tgnd9 iKD ± ATc. 7 dpi lysis plaque areas were measured. No plaques were observed for Tgnd9 iKD +ATc. Means of plaque area ± SD of 3 biological samples. Representative of 3 independent assays. b, Intracellular replication. Left: ΔKu80-Tir1 (Ctrl) and Tgnd6 iKD ± IAA. Right: ΔKu80-TaTi (Ctrl) and Tgnd9 iKD ± ATc. The percentage of vacuoles containing 2, 4, 8, 16 or 32 parasites was determined for 200 vacuoles. Means ± SD from 3 biological samples. Representative of 3 independent experiments. c, Percentage of egressed vacuoles. Left: ΔKu80-Tir1 (Ctrl) and Tgnd6 iKD ± IAA. Right: ΔKu80-TaTi (Ctrl) and Tgnd9 iKD ± ATc. Egress events were quantified for 200 vacuoles. Means ± SD from 3 biological samples. Representative of 2 independent experiments. d, Conoid protrusion assay. The graph represents the percentage of protruded conoids induced by 5 μM A23187, or DMSO as control. Means ±SD from 3 biological samples. Representative of 3 independent experiments. e, Gliding assays were performed with ΔKu80-Tir1 (Ctrl) and Tgnd6 iKD parasite lines ± IAA 24 h (left) and ΔKu80-TaTi (Ctrl) and Tgnd9 iKD parasite lines ± ATc 72 h (right). Trails were revealed by IFA using anti-SAG1 antibodies. Scale bar=10 µm. Representative of 3 experiments. f, Quantification of parasite motility by time−lapse video microscopy. Data are plotted as a percentage of circular, helical and twirling movements. Values represent mean of 2–4 movies. g, Quantification of attachment on glutaraldehyde fixed host cell. As control of microneme secretion-dependent attachment, parasites were treated with BAPTA-AM. h, Quantification of rhoptry secretion by e-vacuoles assay. Tgnd6 iKD was compared to the background line Δku80-Tir1 ± IAA 24 h and Tgnd9 iKD was compared to the background line Δku80-TATi ± Atc 72 h. (g,h) Means ± SD from 3 biological samples. Representative of one experiment. The significance of the results was assessed using a parametric paired t-test (Student’s two-tailed t-test). ns = non-significant.
