Figure 2

Effect of an inducible Pfubc13 knockout in P. falciparum on parasite growth and ubiquitin ubiquitylation (a) Growth over two cycles of intraerythrocytic development for parasites treated with either DMSO (control) or rapamycin to induce Di-Cre mediated excision within ubc13. A synchronised parasite population (0.2% parasitemia) was treated with either DMSO or rapamycin for 24 h; parasitemia was measured by FACS analysis. Data are shown as mean ± standard error of the mean (SEM) from triplicate experiments performed in duplicate. (b) Analysis of parasites by light microscopy of Giemsa-stained thin smears, from parasites treated with either DMSO or rapamycin. At 48 h post invasion (PI), schizonts and ring stages were visible in DMSO treated cultures and there were fewer ring stages in the rapamycin treated cultures. By 96 h PI, second cycle ring stages were visible in DMSO-treated cultures and morphologically abnormal late stage parasites were predominant in rapamycin-treated cultures. Scale bar is 5 µm. (c) Parasitemia over up to 5 cycles of intraerythrocytic growth. The control (DMSO-treated) parasites were monitored for only two cycles; starting parasitemia, 0.3%. The rapamycin-treated parasites were monitored over five cycles; starting parasitema, 0.3 and 1%. The data shown are mean ± SEM from duplicate experiments. (d) The parasite populations were monitored by PCR amplification with primers P1/P4 (see Supplementary Fig. 1) at cycle 3 and cycle 5; no parasites without excision were detected in the rapamycin treated sample. (e) Analysis of schizont lysates by immunoblotting with antibodies specific for either Lys48 or Lys63 ubiquitylation of ubiquitin. Equal amounts of protein from rapamycin-treated or –untreated parasites at 44 h PI were resolved by SDS-PAGE, transferred to nitrocellulose and then probed with antibodies specific for either K48 or K63 ubiquitin linkages, or antibodies specific for BiP as a loading control. Full length gels and immunoblots are presented in Supplementary Figs. 3 and 4.