Figure legends
Fig. 1. Localization of P. berghei Scd in the parasite endoplasmic reticulum. (A ) IFA with asexual blood stage parasites confirmed Scd expression, which colocalized with the endoplasmic reticulum (ER) marker Bip. (B ) The expression of Scd was monitored during liver stage development at different time points, and we found late expression of Scd at 48 and 55 hpi, which colocalized with the ER marker BiP. The nuclei were stained with Hoechst.
Fig. 2. Scd KO parasites display defects in late liver stage development. (A ) To determine the liver parasite burden, sporozoites were injected intravenously into C57BL/6 mice, livers were homogenized at 36, 55 and 72 hpi, RNA was isolated, and transcripts were quantified using real-time PCR. The 18S rRNA copy number was determined and normalized to mouse GAPDH transcripts. TheP. berghei 18S rRNA copy number was comparable in WT GFP andScd KO parasites at 36 hpi but significantly lower in ScdKO parasites than in WT GFP parasites at 55 hpi, and at 72 hpi, it was opposite and found to be significantly higher in KO parasites, indicating that only WT GFP parasites were able to egress from the liver. Similar results were obtained in three independent experiments. Data represent the mean ± SD, n = 5 mice per group. (B ) A lower level of MSP1 transcripts at 55 hpi in KO-infected liver suggests a failure in the maturation of parasites. Data are presented as the mean ± SEM from two independent experiments (significant difference (p=0.0132), Student’s t test).
Fig. 3. Scd KO liver stages grow normally. (A) Sporozoite-infected HepG2 cells were fixed at 12, 24, 36, 48, and 62 hpi and immunostained with anti-UIS4 antibody. Nuclei were stained with Hoechst. Visually, liver stage growth appeared to be comparable inScd KO and WT GFP parasites. (B, D and F ) The EEF numbers were found to be normal at 36, 48, and 62 hpi (p=0.3950 at 36 hpi, p= 0.9419 at 48 hpi, and p= 0.6070 at 62 hpi). (C, E and G ) Determination of EEF size at 36, 48, and 62 hpi. The size of EEFs was comparable between WT GFP and Scd KO parasites (p=0.5096 at 36 hpi, p=0.6269 at 48 hpi, and p= 0.1642 at 62 hpi). The data were pooled from three independent experiments. Data represent the mean ± SD. P values were determined by an unpaired, two-tailed Student’s t test.
Fig. 4. Scd KO parasites arrest late during liver stage development and do not form hepatic merozoites.(A ) Infected HepG2 cells were harvested at 62 hpi, fixed, and immunostained with anti-merozoite surface protein 1 (MSP1) antibody, and DNA was stained with Hoechst. We found normal segregation of nuclei and formation of merozoites in WT GFP, but it was impaired in Scd KO parasites. (B ) Counting of nuclei in the EEF revealed impaired nuclear division in KO parasites (p<0.001, Student’s t test). The data were pooled from three independent experiments. Data represent the mean ± SD. (C ) The merosome numbers were counted using a hemocytometer. Scd KO parasites formed EEFs that were comparable to WT GFP parasites but failed to form merosomes, a significant difference (p<0.0001, Student’s t test).
Fig. 5. Lack of Scd leads to defects in organelle morphology and biogenesis. (A ) Infected HepG2 cells were fixed at 62 hpi and immunostained with apicoplast marker anti-ACP antibody. WT GFP showed normal branching of the apicoplast, which was impaired in Scd KO parasites. (B ) Immunostaining of EEFs with an ER marker anti-Bip antibody revealed impaired branching in Scd KO parasites.