Figure 3

Deletion of Pfubc13 renders parasites more susceptible to the mutagen, methyl methanesulfonate (MMS) and dihydroartemisinin (DHA), but not chloroquine (CQ). (a) Synchronised parasite populations (5 to 8 h post invasion [PI], 1% parasitemia and 3% hematocrit) were pre-treated with DMSO (control) or rapamycin to induce ubc13 excision for 24 h, and then treated with 2.25 mM or 4.5 mM MMS for 0, 20, 40, 60 and 100 min. Parasite survival was assessed at 48 h. The data shown are mean ± SEM from duplicate experiments. (b) Long term survival of parasites treated with MMS. Synchronized parasite populations (5 h PI, 0.5% parasitemia and 3% hematocrit) were treated with rapamycin or DMSO and with or without 500 µM MMS. The cells were then washed to remove the drugs 48 h after the start of the experiment and the incubation was continued in complete medium. Giemsa-stained thin blood smears were examined at the time points indicated to determine percentage parasitemia. The data shown are mean ± SEM from triplicate experiments. (c) Parasites were treated with rapamycin or DMSO and increasing concentrations of either DHA or CQ. The percentage parasite survival was measured, allowing the EC₅₀ of the drugs in each condition to be calculated. The data shown are mean ± SEM from triplicate experiments The DHA EC₅₀ was 1.69 nM (95% CI: 1.41 to 1.99) and 4.1 nM (95% CI: 3.63 to 4.63) for the rapamycin and DMSO treated parasites, respectively, which is significantly different (P < 0.005, t-test), whereas there was no significant difference in the CQ EC₅₀ (18.51 [95% CI: 16.05 to 20.20] and 20.19 [95% CI: 18.53 to 21.48]) for the two treatments.