Figure 6. PfLARC2 parasites display severe defects in late liver stage differentiation.

(A) The schematic depicts the experimental design. To evaluate LS development of PfLARC2 in FRG NOD huHep mice, three million aseptic, cryopreserved sporozoites of either PfNF54-WT parent strain (PfSPZ-WT) and three million PfSPZ-LARC2 were injected intravenously into two FRG NOD huHep mice per group, respectively. Livers were harvested on days 6 and 7 post infection, fixed and liver tissue sections used for IFA analysis. (B) Comparison of PfSPZ-WT and PfSPZ-LARC2 LS parasite infection density at 6 days post infection. Liver stages were counted from sections of four liver lobes of mice infected with either PfSPZ-WT or PfSPZ-LARC2 within an approximate total area of 500 mm² of tissue using CSP and Exp1 antibodies as parasite markers. There is no significant difference in LS infection density between PfSPZ-WT and PfSPZ-LARC2. Data is represented as mean ± SD. Each datapoint refers to the number of LS parasites per cm² of four liver lobes of mice infected with either PfSPZ-WT or PfSPZ-LARC2. Statistical analysis was carried out using unpaired t-test. P > 0.05 is taken as ns. (C) Comparison of the size of LS parasites (based on area at the parasite’s largest circumference) between PfSPZ-WT and PfSPZ-LARC2 at 6- and 7-days post infection. There is no significant difference in LS size between PfSPZ-WT and PfSPZ-LARC2. Data is represented as mean ± SD. Each datapoint refers to the mean size of at least 25 parasites for each timepoint. Statistical analysis was carried out using two-way ANOVA using Tukey’s multiple comparison test. P > 0.05 is taken as ns. Liver stage development and differentiation was compared between PfSPZ-WT and PfSPZ-LARC2 at 7 days post infection using antibodies against; (D) the PPM protein CSP (green), the PVM protein Exp1 (red), and, mature LS stage merozoite markers (E) MSP1 and (F) mTIP on day 7 post infections. DNA is stained with DAPI. Scale bar is 20 µm. PfSPZ-LARC2 late LS schizonts display aberrant distribution of CSP in late LS schizonts, in contrast to in PFSPZ-WT, where CSP is localized to the PPM and delineates cytomeres. No PfSPZ-LARC2 LS merozoite formation was observed. MSP-1 expression was very weak and there was a complete lack of expression of mTIP. In contrast, PfSPZ-WT LS expressed both MSP1 and mTIP and these proteins delineated LS stage merozoites. Source data are available online for this figure.