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.H uman malaria parasites exhibit a complex life cycle consisting of asexual phases within human hepatocytes and erythrocytes, with the latter directly responsible for disease manifestations. Within red blood cells, these parasites can also undergo gametocytogenesis, a process during which they interrupt their asexual replication and differentiate to form morphologically and functionally distinct sexual-stage gametocytes (1). These sexual forms serve as precursors for male and female gametes, which develop in the mosquito where they undergo mating, meiosis and several mitotic cycles to produce sporozoites. In Plasmodium falciparum, the causative agent of the most severe form of human malaria, the progression from immature stage I to mature stage V gametocytes takes ∼10 d (2). However, the biological processes that regulate gametocytogenesis remain unknown. Thorough understanding of these processes is crucial to the development of a new generation of dual activity antimalarials that can inhibit both infection and transmission.Phosphatidylcholine (PC), the predominant phospholipid produced by malaria parasites, plays essential structural and regulatory roles in parasite development and differentiation (reviewed in ref.3). Lipid metabolic and genetic studies in P. falciparum have demonstrated the presence of two pathways for PC biosynthesis (Fig. S1): ...

Probing Plasmodium falciparum sexual commitment at the single-cell level

“…LysoPC uptake is drastically reduced in gametocytes 16 , suggesting that PMT activation is both a general response to nutrient depletion and preparation for increased requirement of this alternative substrate arm during gametocyte development. Indeed, previous work has demonstrated that PMT is essential for gametocyte maturation and mosquito infection 30, 31 .…”

An Alternative Mechanism for the Methylation of Phosphoethanolamine Catalyzed by Plasmodium falciparum Phosphoethanolamine Methyltransferase*

“…Enzyme Assays and Isothermal Titration Calorimetry-Standard assay conditions for PMT activity were 0.1 M Hepes-KOH (pH 8), 2 mM Na 2 EDTA, 10% glycerol, 2.5 mM AdoMet (100 nCi of [methyl- 14 C]AdoMet), and 5 mM phosphobase substrate (either pEA or pDME) in 100 l. Protein (10 g) was incubated for 10 min at room temperature with reactions terminated and product quantified as described previously (8,9). Steady-state kinetic analysis of wild-type and mutant PfPMT was performed as described previously (8,9,18).…”

“…PfPMT plays an important role in the metabolism of the malaria parasite by supporting the synthesis of phospholipids during reproduction and growth. Disruption of the PfPMT gene leads to a reduced phosphatidylcholine synthesis from serine, growth and survival defects, and the loss of parasite transmission to mosquitoes (12)(13)(14). Because of its critical role in Plasmodium and the lack of PMT homologs in mammals, PfPMT, along with other enzymes in phospholipid metabolism, is a target for antiparasitic drug development (5,6,(15)(16)(17).…”

Sex-Specific Biology of the Human Malaria Parasite Revealed from the Proteomes of Mature Male and Female Gametocytes

“…More proteins associated lipid metabolism is in agreement with a significant increase and compositional change of lipids in gametocytes (43,44). Of them, three signal lipidassociated proteins (phosphoethanolamine methyltransferase, cytidine diphosphate-diacylglycerol synthase and calponin homology domain-containing protein) have been studied, showing that the disruption of these genes resulted in the arrest of gametocyte development (37,80). The mechanisms underlining these biological disparities between these Plasmodium species remain to be further elucidated.…”

“…Another member of AP2 family, AP2-G2, may regulate gametocyte development by repression of specific transcripts for asexual and stages beyond gametocyte development (26). In order to prepare for rapid changes and subsequent development in free-living environment in mosquito, gametocytes adopt several strategies including storage of "maternal" mRNAs by the DOZI and possibly Puf protein complexes that can be mobilized rapidly upon stage transition (27)(28)(29)(30)(31), increased expression of protein kinases and phosphatases responsible for signal transduction (32)(33)(34)(35)(36)(37)(38), and activation of tricarboxylic acid cycle and lipid metabolism for energy and lipid requirements (39 -44).…”