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.