Ubiquitin activation is essential for schizont maturation in Plasmodium falciparum blood-stage development
Fig 2
Treatment with MLN7243 arrests parasites at the schizont stage and blocks protein ubiquitylation.
(A) Parasites were treated with either MLN7243 or DMSO carrier, stained with Hoechst dye and analysed by FACS at three time points: 3, 44, and 64 h post invasion, shown in red, blue and orange respectively. Haploid ring stages transition to multinucleate schizonts (shown at 44 h) and in the control DMSO-treated cultures, merozoite reinvasion produces new haploid intraerythrocytic parasites (shown at 64 h). The MLN7243-treated parasites do not release merozoites or form new haploid intraerythrocytic parasites. (B) Microscopy of Giemsa-stained parasites at 24, 44 and 52 h post invasion, showing that both cultures develop into schizonts but by 52 h control parasites have formed new ring stages and the MLN7243-treated parasites remain as schizonts. (C) The percentage of parasitemia of the samples analysed in Panel A (mean +/- standard deviation of technical triplicates) in culture at 3, 44 and 64 h post infection measured by FACS analysis. During intracellular development there is no change in parasitemia, but by 64 h the parasitemia has increased in the control (DMSO-treated) and not the MLN7243-treated culture. (D) Protein ubiquitylation in schizonts either treated (+) or untreated (-) with MLN7243 during development from ring stages. Western blots were probed with a ubiquitin-specific antibody or antibodies specific for K48- and K63-linked polyubiquitylation. Antibodies to parasite GAPDH were used as a loading control. The size of molecular mass markers is shown on the left side of the panel.