SUMMARY Malaria parasites scavenge nutrients from their host but also harbor enzymatic pathways for de novo macromolecule synthesis. One such pathway is apicoplast-targeted type II fatty acid synthesis, which is essential for late liver stage development in rodent malaria. It is likely that fatty acids synthesized in the apicoplast are ultimately incorporated into membrane phospholipids necessary for exoerythrocytic merozoite formation. We hypothesized that these synthesized fatty acids are being utilized for apicoplast-targeted phosphatidic acid synthesis, the phospholipid precursor. Phosphatidic acid is typically synthesized in a three-step reaction utilizing three enzymes: glycerol 3-phosphate dehydrogenase, glycerol 3-phosphate acyltransferase and lysophosphatidic acid acyltransferase. The Plasmodium genome is predicted to harbor genes for both apicoplast- and cytosol/endoplasmic reticulum-targeted phosphatidic synthesis. Our research shows that apicoplast-targeted P. yoelii glycerol 3-phosphate dehydrogenase and glycerol 3-phosphate acyltransferase are expressed only during liver stage development and deletion of the encoding genes resulted in late liver stage growth arrest and lack of merozoite differentiation. However, the predicted apicoplast-targeted lysophosphatidic acid acyltransferase gene was refractory to deletion and was expressed solely in the endoplasmic reticulum throughout the parasite lifecycle. Our results suggest that P. yoelii has an incomplete apicoplast-targeted phosphatidic acid synthesis pathway that is essential for liver stage maturation.

Plasmodium falciparum(Pf) is causing the greatest malaria burden, yet the liver stages (LS) of this most important parasite species have remained poorly studied. Here, we used a human liver-chimeric mouse model in combination with a novel fluorescentPfNF54 parasite line (PfNF54cspGFP) to isolatePfLS-infected hepatocytes and generate transcriptomes that cover the major LS developmental phases in human hepatocytes. RNA-seq analysis of earlyPfLS trophozoites two days after infection, revealed a central role of translational regulation in the transformation of the extracellular invasive sporozoite into intracellular LS. The developmental time course gene expression analysis indicated that fatty acid biosynthesis, isoprenoid biosynthesis and iron metabolism are sustaining LS development along with amino acid metabolism and biosynthesis. Countering oxidative stress appears to play an important role during intrahepatic LS development. Furthermore, we observed expression of the variant PfEMP1 antigen-encoding var genes, and we confirmed expression of PfEMP1 protein during LS development. Transcriptome comparison of the latePfliver stage schizonts withP. vivax(Pv) late liver stages revealed highly conserved gene expression profiles among orthologous genes. A notable difference however was the expression of genes regulating sexual stage commitment. WhilePvschizonts expressed markers of sexual commitment, thePfLS parasites were not sexually committed and showed expression of gametocytogenesis repression factors. Our results provide the first comprehensive gene expression profile of the human malaria parasitePfLS isolated duringin vivointrahepatocytic development. This data will inform biological studies and the search for effective intervention strategies that can prevent infection.