Ubiquitin activation is essential for schizont maturation in Plasmodium falciparum blood-stage development
Fig 5
Generation of a parasite line with a rapamycin-inducible uba1 gene knockout and its response to treatment with either rapamycin, MLN7243, or DMSO.
(A) CRISPR Cas9 was used to modify the 3' end of the uba1 gene in the II-3 parasite line, inserting loxPint in place of the last intron, a recodonised coding sequence fused to GFP, a second loxPint sequence and sequence coding for a triple HA tag. The parasite has a floxed gene from which a UBA1-GFP fusion protein is expressed. In the presence of rapamycin the floxed sequence is excised to produce an inactive truncated UBA1 protein. (B) Diagnostic PCR with primers designed to detect successful integration of the floxed sequence and its excision in the presence of rapamycin. DNA from the parental (II-3) and three cloned recombinant lines (G107, G109 and G111), following growth with or without rapamycin, was amplified using the primers 121 and HArev. Amplified bands of 1.8 kb and 0.74 kb were expected from the floxed and excised (+rapamycin) locus in the progeny parasites and no product was expected from the parental II-3. (C) Western blot with anti-GFP antibodies probed against schizont extracts of parasites (clones G107, G109 and G111) with the floxed and excised (+ rapamycin) uba1 locus. Reactivity of antibodies to BiP (Binding immunoglobulin Protein, GRP-78) was used as a loading control. (D) Growth of parental (II-3) and two independent recombinant parasite clones (G109 and G111) treated at the early ring-stage with either DMSO (control) or 20 nM rapamycin for 24 h and then stained with Hoechst dye and analysed by FACS 48 h later. The data are presented as the mean growth as a percentage of the DMSO-treated samples in triplicate samples from three independent experiments. (E) G109 parasites were treated at the early ring stage with either DMSO, rapamycin or MLN7243 and the parasitemia measured at specific time points after invasion following staining with Hoechst dye and FACS analysis. At 48h the parasites were washed to remove drug and culture was continued for a further 12 h. Data are shown as mean ± standard deviation of technical triplicates. Genetic truncation or inhibition of UBA1 blocked parasite growth. (F) G109 parasites were treated at the early ring stage with either DMSO, rapamycin or MLN7243 and the number of nuclei in individual parasites was counted at specific time points after invasion following staining with Hoechst dye and fluorescence microscopy. Shown is the median with the 95% confidence interval. One way ANOVA with Sidak’s multiple comparison test: **** DMSO v. RAPA, p < 0.0001, DMSO v. MLN7243, p < 0.0001; nsRAPA v. MLN7243 p = 0.937. (G) Immunofluorescence microscopy images of G109 parasites treated at the early ring stage with either DMSO or rapamycin and stained with antibodies to either GAP45 or ARO at 45 h after invasion. GAP45 is a component of the inner membrane complex (IMC) and ARO is a rhoptry protein. Merged images with DAPI staining and differential interference contrast (DIC) are included. Scale bar is 2 micron.