A pathway for phosphatidylcholine biosynthesis in Plasmodium falciparum involving phosphoethanolamine methylation

Abstract

Plasmodium falciparum is the causative agent of the most severe form of human malaria. The rapid multiplication of the parasite within human erythrocytes requires an active production of new membranes. Phosphatidylcholine is the most abundant phospholipid in Plasmodium membranes, and the pathways leading to its synthesis are attractive targets for chemotherapy. In addition to its synthesis from choline, phosphatidylcholine is synthesized from serine via an unknown pathway. Serine, which is actively transported by Plasmodium from human serum and readily available in the parasite, is subsequently converted into phosphoethanolamine. Here, we describe in P. falciparum a plant-like S-adenosyl-l-methionine-dependent three-step methylation reaction that converts phosphoethanolamine into phosphocholine, a precursor for the synthesis of phosphatidylcholine. We have identified the gene, PfPMT, encoding this activity and shown that its product is an unusual phosphoethanolamine methyltransferase with no human homologs. P. falciparum phosphoethanolamine methyltransferase (Pfpmt) is a monopartite enzyme with a single catalytic domain that is responsible for the three-step methylation reaction. Interestingly, Pfpmt activity is inhibited by its product phosphocholine and by the phosphocholine analog, miltefosine. We show that miltefosine can also inhibit parasite proliferation within human erythrocytes. The importance of this enzyme in P. falciparum membrane biogenesis makes it a potential target for malaria chemotherapy.

Fig. 1.

Evidence for a PEAMT (Pfpmt) activity in P. falciparum. (A) TLC analysis of the extracted lipid (i) and aqueous (ii) phase after labeling of P. falciparum infected erythrocytes with [¹⁴C]-Etn. The identity of phospholipids (i) and aqueous compounds (ii) was determined by using appropriate standards. *, the theoretical position of choline. (B) Pfpmt activity in P. falciparum extracts using 100 μM P-Etn and 100 μM SAM as substrate and cosubstrate, respectively. The activity was measured at 37°C in the absence (-) or presence (+) of 600 μg of P. falciparum protein extract, and the product P-Cho was purified by using a AG-50(H⁺) ion-exchange resin. Each value is the mean ± SD of duplicate experiments. (C) TLC analysis of the reaction product when reaction was performed in the absence (-) or presence (+) of 600 μg of P. falciparum protein extract; lane S1, standard [¹⁴C]P-Cho. The origin of the migration is indicated.

Fig. 2.

Expression and sequence analysis of Pfpmt. (A) Quantification of PfPMT expression by real-time PCR analysis in ring (R), trophozoite (T), and schizont (S) stages. The indicated fold change in PfPMT transcription was calculated relative to the ring stage expression. Data were normalized to expressed level of the seryl-tRNA synthetase encoding gene. The standard deviation of two independent experiments is given. (B) Sequence alignment of the polypeptide sequence encompassing the PEAMT catalytic motifs (I, p-I, II, and III) from P. falciparum (Pfpmt; AN AY429590) and S. oleracea (So-Nt and So-Ct, AN AF237633), Arabidopsis thaliana (At-Nt and At-Ct, AN AAG41121) PEAMT, C. elegans (Cepmt; AN AAB04824), and A. gambiae (Agpmt). Sequence identity is indicated in dark gray, and similarity is indicated in light gray. (C) Schematic representation of the bipartite structure of plant PEAMTs and monopartite structures of Pfpmt, Cepmt, and Agpmt. The four motifs (I, p-I, II, and III) of each PEAMT catalytic domain(s) are indicated as black boxes. The size of the protein in amino acid (aa) is indicated.

Fig. 3.

Recombinant Pfpmt specifically catalyzes the methylation of P-Etn into P-Cho. (A) Time course of Pfpmt SAM-dependent methylation of 100 μM P-Etn at 37°C (•) and 0°C (▪) using 10 μg of recombinant Pfpmt. The reaction was performed as described in Materials and Methods. (Inset) A TLC analysis of the product of the Pfpmt reaction after a 30-min incubation at 37°C (lane 1) and 0°C (lane 2); lane S1, standard [¹⁴C]P-Cho. (B) Substrate specificity of Pfpmt in the presence (+) or absence (-) of 100 μM P-Etn, Etn, and PtdEtn. Each datum represents an average of a duplicate ± SD.

Fig. 4.

Kinetics of Pfpmt reaction using recombinant enzyme. Pfpmt activity was measured in the presence of varying concentrations of P-Etn (A) and SAM (B). In A, SAM was present at a concentration of 2 mM, whereas in B, P-Etn was added at a final concentration of 3 mM. The reaction was carried out as described in Materials and Methods. The formation of P-Cho was quantified per mg of protein per minute. The curve for velocity (V) versus substrate concentration ([S]) was fit to the Michaelis–Menten equation (V = V_max × S/[K_m + S]). Each datum point represents an average of a duplicate ± SD.

Fig. 5.

Inhibition of recombinant Pfpmt activity and P. falciparum growth. (A) Inhibition of Pfpmt activity by P-Cho. Inhibition assay was performed by adding various concentrations of P-Cho (0–500 μM) as described in Materials and Methods. (B) Inhibition of Pfpmt activity by 50 μM P-Cho, 50 μM adenosyl-l-homocysteine (AdoHcy), and 1, 10, 50, and 100 μM miltefosine. Control, Pfpmt activity without inhibitor. (C) Effect of several concentrations of miltefosine on [³H]hypoxanthine incorporation in cultured parasites. Each value is the mean ± SD of at least duplicate experiments.

Fig. 6.

Model of the pathways for PtdCho biosynthesis in P. falciparum. Plant-like reactions are indicated as dotted lines, and the new identified pathway is shown in gray. DAG, diacylglycerol; PtdIno, phosphatidylinositol; PfPSS, phosphatidylserine synthase; PfPSD, phosphatidylserine decarboxylase; PfEK and PfCK, ethanolamine and choline kinases; PfECT and PfCCT, P-Etn and P-Cho cytidyltransferases; PfEPT and PfCPT, ethanolamine and choline phosphotransferases; PfSD, serine decarboxylase.