Plasmodium falciparum phosphoethanolamine methyltransferase is essential for malaria transmission

Abstract

Efficient transmission of Plasmodium species between humans and Anopheles mosquitoes is a major contributor to the global burden of malaria. Gametocytogenesis, the process by which parasites switch from asexual replication within human erythrocytes to produce male and female gametocytes, is a critical step in malaria transmission and Plasmodium genetic diversity. Nothing is known about the pathways that regulate gametocytogenesis and only few of the current drugs that inhibit asexual replication are also capable of inhibiting gametocyte development and blocking malaria transmission. Here we provide genetic and pharmacological evidence indicating that the pathway for synthesis of phosphatidylcholine in Plasmodium falciparum membranes from host serine is essential for parasite gametocytogenesis and malaria transmission. Parasites lacking the phosphoethanolamine N-methyltransferase enzyme, which catalyzes the limiting step in this pathway, are severely altered in gametocyte development, are incapable of producing mature-stage gametocytes, and are not transmitted to mosquitoes. Chemical screening identified 11 inhibitors of phosphoethanolamine N-methyltransferase that block parasite intraerythrocytic asexual replication and gametocyte differentiation in the low micromolar range. Kinetic studies in vitro as well as functional complementation assays and lipid metabolic analyses in vivo on the most promising inhibitor NSC-158011 further demonstrated the specificity of inhibition. These studies set the stage for further optimization of NSC-158011 for development of a class of dual activity antimalarials to block both intraerythrocytic asexual replication and gametocytogenesis.

Fig. 1.

PfPMT expression during gametocyte development. (A) Wild-type 3D7 parasites were precultured at 2% parasitemia and 6% hematocrit in complete medium and maintained at 37 °C until the cultures reached 10% parasitemia. Parasites were then transferred to GM culture conditions and samples collected over time. Expression of PfPMT and the gametocyte-specific protein Pfg27 in gametocyte stages I, II, III, IV and V was monitored by immunofluoresence analysis using antibodies directed to PfPMT (green) or Pfg27 (red). Areas of overlap between PfPMT and Pfg27 appear in yellow. Nuclear staining was achieved using Hoechst 33258 (blue). (B) Culture sample showing adjacent trophozoite (T)- and gametocyte (G)-infected erythrocytes stained with anti-PfPMT and anti-Pfg27 antibodies.

Fig. 2.

Parasites lacking PfPMT are altered in sexual differentiation and transmission. (A) Gametocytemia expressed as percent parasitemia at different days of culture in GM culture conditions for wild-type 3D7, pfpmtΔ+PfPMT and pfpmtΔ mutant strains (black, white, and gray columns, respectively). Data are means ± SDs of triplicate assays. (B) Wild-type (3D7) and pfpmtΔ parasites were precultured at 2% parasitemia and 6% hematocrit in complete medium and maintained at 37 °C until the cultures reached 10% parasitemia in the absence or presence of the CDP-choline precursor choline (20 µM). Parasites were then maintained under GM culture conditions in the absence or presence of 20 µM choline, and the total number of mature gametocytes (IV and V) was determined at day 11. For each condition, a total of 5,000 erythrocytes were counted. (C) Giemsa-stained thin blood smears of gametocyte stages detected in wild-type, pfpmtΔ, and complemented pfpmtΔ+PfPMT parasites. No stage-IV or -V gametocytes could be detected in the knockout strain. (D) Immunofluorescence analysis of wild-type, pfpmtΔ and complemented pfpmtΔ+PfPMT parasites expressing PfPMT under the regulatory control of the P. falciparum CAM1 promoter (9). PfPMT (green), Pfg27 (red), and Hoechst (blue). Error bars indicate SD of light microscopy counts from three independent experiments. *P < 0.05. (E) Phase-contrast images of a wild-type P. falciparum-infected mosquito midgut. Arrows indicate individual P. falciparum 3D7 oocysts. No oocysts could be detected in the midguts of mosquitoes fed on pfpmtΔ-infected red blood cells. Both wild-type and pfpmtΔ parasites were maintained under GM conditions before mosquito feeding. (F) Quantitative PCR results from Anopheles stephensi mosquitoes fed on either wild-type or pfpmt∆ cultures. Each dot indicates individual mosquitoes that were harvested 8 d after artificial blood feeding. Dots on the x axis indicate mosquitoes from which no P. falciparum 18S rRNA could be detected.

Fig. 3.

Inhibition of gametocyte development and maturation by PfPMT inhibitors. (A and C) Diagrams of the experimental plan to determine luciferase activity immediately following removal of the compound (A) or on day 16 (C). (B and D) Heat maps representing the effect of PfPMT inhibitors on gametocyte development as measured by luciferase activity in the transgenic line NF54-pfs16-GFP-LUC. Parasites were treated for 48 h (blue bar) with 10 µM of the indicated compound at day 5, 8, or 12 following parasite inoculation. The antimalarial artemisinin (ART) was used as a control at the same concentration. (B) Luciferase activity was measured 48 h posttreatment. (D) Luciferase activity was measured at day 16. (E–G) inhibition of early (E), intermediate (F), and late (G) gametocyte development by artemisinin (A) and NSC-158011 (N). Results are the mean ± SEM of triplicate from three independent assays. Each figure is a representative of one of three independent experiments.

Fig. 4.

Biochemical and genetic evidence for specific inhibition of PfPMT by NSC-158011. (A) Michaelis–Menten representation of PfPMT activity in the absence or presence of increasing concentrations of NSC-158011 and changing concentrations of the substrate (P-Etn). The lines shown represent the global fit of all data to the nonlinear fit of Michaelis–Menten mixed model of inhibition. The chemical structure of NSC-158011 is represented. (B and C) The pem1∆pem2∆ strains with pYES2.1-PfPMT vector (B) and wild-type strains with empty vector (C) were inoculated into uracil dropout synthetic galactose medium supplemented with 10 μM ethanolamine (Etn) and grown overnight. Cells were harvested and reinoculated at an A₆₀₀ = 0.005 in uracil dropout synthetic galactose medium supplemented with 100 μM ethanolamine and/or 100 μM choline (Cho), in the absence or presence of 25 μM NSC-158011 as indicated. Cell growth at 30 °C was monitored by A₆₀₀. (D and E) Labeled phospholipids were extracted from pem1∆pem2∆ strains carrying pYES2.1-PfPMT vector grown for 17 h in synthetic medium containing 8 µCi of [³³P] orthophosphoric acid. The lipid classes were resolved by TLC, visualized by PhosphorImager (D), and quantified by liquid scintillation spectrometry (E). Data are the means ± SD for four experiments.