Fig. 2: Conditional knockout of PfRh5, PfPTRAMP and PfCSS shows their function was essential for invasion of human erythrocytes by P. falciparum merozoites.

a, Quantitation of merozoite invasion of erythrocytes when expression of PfRh5, PfPTRAMP or PfCSS was knocked down using rapamycin. Shown are each P. falciparum parasite line in which either PfRh5 (3D7–PfRh5iKO), PfPTRAMP (3D7–PTRAMPiKO) or PfCSS (3D7–CSSiKO) is under inducible knockout control with rapamycin (R) compared with control with no rapamycin (C). Histogram represents two independent experiments (Expt 1 and Expt 2) of each parasite line with mean ± s.e.m. b, Representative snapshots showing parasites (cyan) interacting with erythrocytes (magenta) pre-loaded with Ca²⁺ indicator (yellow) displayed using Imaris in 3D Blend mode and XZ views. In the control condition (top; 3D7–PfRh5iKO), the parasite shows a Ca²⁺ flux with internalization and echinocytosis. In rapamycin-treated (Rapa) (bottom; 3D7–PfRh5iKO), the parasite caused deformations on the erythrocyte but no invasion or echinocytosis. Scale bars, 2 μm. c, PAM time plot showing the first-minute interaction by invading control (blue, n = 32), non-invading control (orange, n = 31) and Rapa-treated parasites (red, n = 41) with neighbouring erythrocytes after parasite egress, where t = 0 represents the timepoint immediately before interaction began. Solid lines represent mean values, and shaded regions represent ±95% confidence interval (CI). d, PAM time plot from two parasite–erythrocyte interactions showing the definition of deformation (solid red and blue lines), which exclude the internalization period and beyond (dashed blue line), as well as periods where PAM ≤ 20 voxels per μm² (dashed red lines). e, PAM time plot from c labelled with thresholds for defining the degree of parasite–erythrocyte interaction and respective images for visualization. Images show three examples in 3D Blend mode view and one example in XY, YZ and XZ views each for weak, moderate and strong deformations, displayed with Imaris. Scale bars, 2 μm. f–h, Bar graphs showing maximum deformation (*P = 0.0308) (f), average deformation (P values from left to right: **P = 0.0043, *P = 0.0268, *P = 0.0197, *P = 0.0137) (g) and total deformation time (P values from left to right: **P = 0.0051, *P = 0.0233, ****P < 0.0001, *P = 0.0155, ****P < 0.0001, ****P < 0.0001) (h) during the first-minute interaction by invading control (blue), non-invading control (orange) and Rapa-treated (red) parasites from 3D7–PfRh5iKO (n = 9 for control, n = 12 for non-invading control, n = 11 for Rapa-treated), 3D7–CSSiKO (n = 11 for control, n = 10 for non-invading control, n = 15 for Rapa-treated) and 3D7–PTRAMPiKO (n = 12 for control, n = 9 for non-invading control, n = 15 for Rapa-treated) parasite lines. Bar heights represent mean values, and error bars represent standard deviation (s.d.). Mann–Whitney two-tailed test.

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