Enzymes involved in plastid‐targeted phosphatidic acid synthesis are essential for Plasmodium yoelii liver‐stage development

Abstract

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.

Fatty acid metabolism in the Plasmodium apicoplast: Drugs, doubts and knockouts

“…Based on genomic data, the Plasmodium apicoplast was initially predicted to possess a three step pathway for phosphatidic acid synthesis using acyl-ACP from FASII and DHAP imported by the pPTs [26] (see Section 2.1). Recent investigation of the pathway in P. yoelii has confirmed the apicoplast localization of its first two enzymes, and demonstrated the activity of the second by complementation [128] (Table 1). However, the enzyme predicted to catalyze the third step in the pathway was instead localized to the ER in P. yoelii, and no alternative capable of fulfilling its function in the apicoplast could be identified [128].…”

“…However, the enzyme predicted to catalyze the third step in the pathway was instead localized to the ER in P. yoelii, and no alternative capable of fulfilling its function in the apicoplast could be identified [128]. These findings suggest the Plasmodium apicoplast lipid precursor synthesis pathway may be incomplete, with the two known enzymes only sufficient to catalyze the conversion of acyl-ACPs and DHAP into the intermediate lysophosphatidic acid [128] (Fig. 2).…”

“…Glycerol-3-phosphate dehydrogenases catalyze the reversible reduction of DHAP to glycerol-3-phosphate using NADH, NADPH or FADH 2 cofactors. Plasmodium parasites possess three G3PDH isoforms, two putatively involved in a glycerol-3-phosphate shuttle in the mitochondrion [170][171][172], and another targeted to the apicoplast [128]. The localization of the P. yoelii apicoplast G3PDH has been confirmed by epitope tagging and visualization in liver stage parasites [128].…”

“…ACP binds to fatty acids and their precursors via its phosphopantetheine prosthetic group, and is essential to shuttle substrates between separate enzymes of the pathway. The apicoplast localization of the P. falciparum and T. gondii ACP have been confirmed by multiple methods [24,25], and both the P. berghei and P. yoelii proteins have been detected using antibodies [97,128]. The function of PfACP has been demonstrated by complementation [129] and recombinant expression in E. coli [65,67], and also through its ability to support FASII enzymes in vitro [65,66].…”

Distinct Prominent Roles for Enzymes of Plasmodium berghei Heme Biosynthesis in Sporozoite and Liver Stage Maturation

“…S2A in the supplemental material), and precise mutations of the two alternative splice products are needed to disentangle the individual roles for parasite propagation. The majority of apicoplast-localized proteins appear to be essential either for blood infection (e.g., key enzymes of [Fe-S] biosynthesis [45]) or during liver stage development (e.g., pyruvate dehydrogenase [PDH] [46], Plasmodiumspecific apicoplast protein important for liver merozoite formation [PALM] [43], and enzymes of phosphatidic acid biosynthesis [47]). Together with FABI and FABB/F of the fatty acid biosynthesis pathway (48), PBGD and UROD are the first apicoplast proteins with distinct essential functions during vector stage development, highlighting the specific roles of apicoplast metabolism at various phases in the complex life cycle.…”