Fig. 1.

PfMyoA is critical for red blood cell invasion by merozoites. a Schematic showing replacement of the wild-type myoa locus with the full-length mutant locus by single crossover recombination in the myoa HR region using a T2A skip peptide (yellow box) to couple genomic integration to neomycin selection. b Treatment of two MyoA-cKO clones (B9 and H6) or WT with rapamycin (RAP) shows an almost complete invasion block (~90%) compared to DMSO treatment (−). This is comparable to the defect with known invasion inhibitors heparin and Cytochalasin D (CytoD) (90-95%). Parasitaemia was measured in the following cycle (cycle 1) by flow cytometry as the percentage of red blood cells (RBCs) that were DNA-positive by staining with SYBR Green I. Mean of three biological replicates (except for WT+heparin: two biological replicates), each three technical replicates, ± standard deviation (S.D.) of biological replicates. Data from each biological replicate were normalized to the DMSO-treated sample for each parasite line. Significance assessed using parametric t-test (paired, two-tailed). c Genotyping PCR of WT or cKO parasites following DMSO (−) or RAP (+) treatment detecting the wild-type (WT, red half-arrow), unexcised (UN, blue half-arrow) and excised (EX, green half-arrow) pfmyoa loci. d Immunofluorescence analysis of cKO parasites following DMSO or RAP treatment. FLAG-tag is detectable in DMSO-treated schizonts fixed ~48 h post-treatment, colocalising with motor complex protein GAP45, while after RAP treatment GFP but not FLAG is detectable, and the signal is restricted to the cytosol, consistent with a non-functional truncated MyoA. Scale bar 1 μm. Image stacks were deconvolved using the EpiDEMIC plugin for Icy, with a z-step size of 200 nm. e Western blot of WT and cKO parasites following DMSO (−) or RAP (+) treatment. Parasites were lysed ~40 h post-treatment, before the end of cycle 0. In DMSO-treated cKO parasites, the original FLAG-tag is detectable, but following RAP-treatment, only GFP is detectable. PfAct1 was used as a loading control. Representative blot shown from three biological replicates