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.