2. 4 Scd KO liver stages grow normally but do not form hepatic merozoites
To further explore the Scd KO phenotype, HepG2 cells were infected with sporozoites, and the culture was fixed at different time points and stained with anti-UIS4 antibody and Hoechst. The ScdKO parasites grew similarly to the WT GFP parasites during liver stage development (Fig. 3A). We counted the EEF numbers and measured the size at 36, 48, and 62 hpi and found comparable numbers and sizes inScd KO and WT GFP parasites (Fig. 3B-G). Next, we checked the formation of merozoites by staining with an anti-MSP1 antibody. WT GFP showed nuclear segregation and merozoite formation, whereas there was abnormal DNA segregation in Scd KO parasites, and merozoite formation was abolished (Fig. 4A). We counted the nuclei in the EEF, which were found to be significantly fewer in Scd KO parasites (Fig. 4B). Next, we observed the culture for the formation of merosomes at 65 hpi and found it only in WT GFP (Fig. 4C). We counted the merosomes in WT GFP, and an equivalent amount of culture supernatant was injected into Swiss mice. The prepatent period was determined by making a Giemsa-stained blood smear. Mice injected with WT GFP merosomes became patent, whereas Scd KO supernatant-injected mice remained negative until the observation period (Table 2). This result suggests that Scd plays an important role in the maturation of hepatic merozoites and is essential for the liver-to-blood stage transition.