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. 2020 Oct 13;9:e60581. doi: 10.7554/eLife.60581

Figure 7. PfELC is essential for parasite invasion of the red blood cell.

(a) Growth curve comparing wildtype (B11) and PfELC-3xHA parasites over the course of three cycles indicates no detrimental effect of genetic modification in the pfelc locus. Mean ring-stage parasitemia is shown of two biological replicates as determined by flow cytometry. (b) Western blot analysis of parasite extracts separated by SDS-PAGE probed with anti-HA and anti-actin antibodies as a loading control. PfELC-3xHA runs at the expected MW of ~21 kDa in mock DMSO -treated samples (D) and the HA signal is lost upon treatment with rapamycin (R), indicating successful excision. (c–d) Representative immunofluorescence assays (IFAs) of schizont and ring-stage parasites co-labeled with the IMC marker GAP45. Peripheral staining is lost after treatment with rapamycin and a diffuse/punctate pattern is seen with anti-cMyc antibodies. (e) R-treated PfELC-3xHA parasites are significantly impaired in invading red blood cells as determined by flow cytometry analysis of ring-stage parasites over 60 hr, comparable to that seen with the non-specific inhibitor heparin. A small amount of residual invasion seen after 48 hr (f) disappears after 96 hr post-treatment (g) suggesting that the absence of PfELC results in complete ablation of invasion. D-treated parasites show no invasion defect. Parasitemias were normalized to D-treated for each line, bars show mean + / - S.D.

Figure 7—source data 1. Source data for parasitology experiments presented in Figure 7.

Figure 7.

Figure 7—figure supplement 1. Genetic integration of LoxP Cre recombinase sites into the Pfelc gene of Plasmodium falciparum.

Figure 7—figure supplement 1.

(a) Schematics of the targeting plasmid (pARL PfELCloxP), expected selection-linked integration (SLI) into the pfelc locus leading to C-terminally tagged PfELC 3xHA and the DiCre-mediated recombinase event resulting in the PfELC conditional KO (cKO). The recodonised version of the pfelc gene is shown as a red striped box, the 3x HA tag is depicted in blue, the T2A skip peptide in gray and the cMyc/FLAG tag in orange. The stop codon is indicated by an asterisk and primers used for genotyping are indicated by numbered arrows. (b) PCR analysis and confirmation of integration into the pfelc locus in transgenic PfELC 3x HA parasites as evidenced by loss of the wild-type band at ~1 kb (P1 and P2) and amplification of an ~1.2 kb (P1 and P3),~1.9 kb (P1 and P4) and ~1 kb (P5 and P2) product. Successful rapamycin-induced excision results in the expected loss of the ~1.2 kb band and size reduction of the ~1.9 kb product leading to PfELC cKO.