2. 3 Scd KO sporozoites infect the liver but fail to establish blood-stage infections in mice
To evaluate the role of Scd in liver stage development, different doses of WT and KO sporozoites were injected intravenously into C57BL/6 mice or infected by mosquito bite, and the appearance of the parasite in the blood was monitored by making a Giemsa blood smear. We found that all the mice injected with WT GFP and Scd comp became patent on day 3, whereas mice inoculated with KO sporozoites remained negative until the observation period (Table 1). The normal prepatent period of Scd comp parasites suggests that the lack of infectivity of KO parasites was due to the deletion of the Scd gene. To determine the stage-specific defect in KO parasites, we first investigated the invasion ability of KO sporozoites. For this, sporozoites were allowed to invade HepG2 cells, and sporozoites present inside and outside of the cells were counted, which revealed normal invasion by KO sporozoites (Fig. S4). Post invasion, sporozoites transform into EEFs that mature into merozoites, which are released 63 to 70 hpi in a controlled manner in the form of merosomes, and then the parasite biomass in the liver declines sharply (Sturm et al. , 2006). To analyze the progress of EEF development in vivo, the livers of infected mice were harvested at 36, 55, and 72 hpi, and parasite biomass was quantified by measuring 18S rRNA copy number using real-time PCR. We found that the 18S rRNA copy number was comparable in WT GFP and KO parasites at 36 hpi, but it was significantly lower at 55 hpi in KO parasites than in WT GFP parasites (Fig. 2A). However, the 18S rRNA copy number at 72 hpi was significantly lower in WT GFP than in KO (Fig. 2A), which suggested that mature WT GFP parasites egress from the liver but that KO parasites failed to mature and remained in the liver. We confirmed the maturation defect by determining the merozoite surface protein 1 (MSP1) transcripts at 55 hpi and found that it was significantly decreased in the KO parasites (Fig. 2B). These data provide evidence that Scd KO parasites failed to mature into hepatic merozoites and did not egress from hepatocytes.