Isoprenoid Precursor Biosynthesis Is the Essential Metabolic Role of the Apicoplast during Gametocytogenesis in Plasmodium falciparum

The malaria parasite harbors a relict plastid called the apicoplast and its discovery opened a new avenue for drug discovery and development due to its unusual, nonmammalian metabolism. The apicoplast is essential during the asexual intraerythrocytic and hepatic stages of the parasite, and there is strong evidence supporting its essential metabolic role during the mosquito stages of the parasite. Supply of the isoprenoid building blocks isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) is the essential metabolic function of the apicoplast during the asexual intraerythrocytic stages. However, the metabolic role of the apicoplast during gametocyte development, the malaria stages transmitted to the mosquito, remains unknown. In this study, we showed that production of IPP for isoprenoid biosynthesis is the essential metabolic function of the apicoplast during gametocytogenesis, by obtaining normal gametocytes lacking the apicoplast when supplemented with IPP. When IPP supplementation was removed early in gametocytogenesis, developmental defects were observed, supporting the essential role of isoprenoids for normal gametocytogenesis. Furthermore, mosquitoes infected with gametocytes lacking the apicoplast developed fewer and smaller oocysts that failed to produce sporozoites. This finding further supports the essential role of the apicoplast in establishing a successful infection in the mosquito vector. Our study supports isoprenoid biosynthesis as a valid drug target for development of malaria transmission-blocking inhibitors.     

Isoprenoid biosynthesis in the erythrocytic stages of Plasmodium falciparum

The development of new drugs is one strategy for malaria control. Biochemical pathways localised in the apicoplast of the parasite, such as the synthesis of isoprenic precursors, are excellent targets because they are different or absent in the human host. Isoprenoids are a large and highly diverse group of natural products with many functions and their synthesis is essential for the parasite's survival. During the last few years, the genes, enzymes, intermediates and mechanisms of this biosynthetic route have been elucidated. In this review, we comment on some aspects of the methylerythritol phosphate pathway and discuss the presence of diverse isoprenic products such as dolichol, ubiquinone, carotenoids, menaquinone and isoprenylated proteins, which are biosynthesised during the intraerythrocytic stages of Plasmodium falciparum.     

Isoprenoid metabolism in Plasmodium falciparum during the intraerythrocytic phase of malaria

Products of the isoprenoid metabolism were identified upon incubations of extracts from Plasmodium falciparum infected red blood cells with [14C] mevalonate. Uninfected erythrocytes and wild type yeast Saccharomyces cerevisiae extracts were used as controls. In parasitized red blood cells as well as in yeast extracts, mevalonate was converted into the biosynthetic isoprenoid precursors of sterol pathway until farnesyl pyrophosphate. In contrast, no mevalonate conversion was observed in uninfected erythrocyte extracts. The isoprenoid metabolism appeared stage-dependent as shown by the increase of radiolabelled farnesyl pyrophosphate amount at the beginning of the schizogonic phase (30-36 hours).     

Phospholipid Biosynthesis in Synchronous Plasmodium falciparum Cultures1

The metabolism of phospholipids in synchronous Plasmodium falciparum-infected erythrocytes was studied over one cycle of 48 h by the incorporation of labeled palmitate, serine, choline, and myo-inositol into cellular lipids. The rates of incorporation of palmitate and serine into total phospholipids and of choline into phosphatidylcholine (PC) were linear with the maturation of the parasite, increasing by a factor of 2–5.6 according to the precursors. The rate of inositol incorporation into phosphatidylinositol was 9.6 times higher at the schizont stage than at the ring stage, with a marked increase in the second half of the cycle. A significant incorporation of palmitate into triglycerides also occurred during the schizont stage of the parasite. The incorporations of serine and palmitate into phosphatidylethanolamine (PE) and PC showed a net increase at approximately the twentieth hour of the cycle, while the radioactivities recovered in phosphatidylserine (PS) had already reached a maximum by this time. These findings indicate an instantaneous transformation of PS into PE and PC through a decarboxylation of PS into PE, then a methylation of PE into PC during the second half of the cycle. Although PS is a minor component of the Plasmodium parasite, our findings demonstrate the important role of this phospholipid as a precursor of PE and PC, which are major constituents of parasite phospholipids.     

Biosynthesis of the 25-kDa protein in the macrogametes/zygotes of Plasmodium falciparum

Synthesis of the 25-kDa protein in the early midgut stages of Plasmodium falciparum was studied, using metabolic inhibitors (colchicine and actinomycin D) and pulse-labeling experiments. Experiments with colchicine showed that, immediately after induction of macrogametogenesis, 25-kDa protein synthesis occurs in both fertilized and nonfertilized macrogametes. The amount of 25-kDa protein synthesized increased slowly during time. Experiments with actinomycin D revealed that the slow increase of synthesis may be dependent on de novo messenger RNA synthesis.     

The Human Malaria Parasite Plasmodium falciparum Is Not Dependent on Host Coenzyme A Biosynthesis

Pantothenate, a precursor of the fundamental enzyme cofactor coenzyme A (CoA), is essential for growth of the intraerythrocytic stage of human and avian malaria parasites. Avian malaria parasites have been reported to be incapable of de novo CoA synthesis and instead salvage CoA from the host erythrocyte; hence, pantothenate is required for CoA biosynthesis within the host cell and not the parasite itself. Whether the same is true of the intraerythrocytic stage of the human malaria parasite, Plasmodium falciparum, remained to be established. In this study we investigated the metabolic fate of [¹⁴C]pantothenate within uninfected and P. falciparum-infected human erythrocytes. We provide evidence consistent with normal human erythrocytes, unlike rat erythrocytes (which have been reported to possess an incomplete CoA biosynthesis pathway), being capable of CoA biosynthesis from pantothenate. We also show that CoA biosynthesis is substantially higher in P. falciparum-infected erythrocytes and that P. falciparum, unlike its avian counterpart, generates most of the CoA synthesized in the infected erythrocyte, presumably necessitated by insufficient CoA biosynthesis in the host erythrocyte. Our data raise the possibility that malaria parasites rationalize their biosynthetic activity depending on the capacity of their host cell to synthesize the metabolites they require.Pantothenate (vitamin B₅) is an essential nutrient for the virulent human malaria parasite Plasmodium falciparum, required to support the rapid growth and replication of the parasite during the intraerythrocytic stage of its life cycle (1–3). In bacteria, plants, and mammalian tissues, pantothenate serves as a precursor of coenzyme A (CoA),³ an essential enzyme cofactor involved in numerous metabolic reactions in the cell. Pantothenate is converted to CoA via five universal enzyme-mediated steps (see Fig. 1).Open in a separate windowFIGURE 1.The CoA biosynthesis pathway.Several decades ago, Trager (4) showed that pantothenate supported the survival of the avian malaria parasite Plasmodium lophurae during its development within duck erythrocytes in vitro. Trager (5, 6) later demonstrated, however, that CoA, and not pantothenate, stimulated exoerythrocytic growth of the intraerythrocytic stage of P. lophurae, and proposed that avian malaria parasites are incapable of metabolizing pantothenate to CoA, and instead rely on CoA synthesized by the host erythrocyte. In support of this proposal, CoA biosynthesis enzymes are readily detectable in duck erythrocytes, but appear to be absent from P. lophurae parasites isolated from their host erythrocyte (7, 8). Pantothenate is therefore required by the P. lophurae-infected duck erythrocyte for CoA biosynthesis within the host cell and not the parasite itself.By contrast with nucleated avian erythrocytes, mammalian erythrocytes are thought to be incapable of CoA biosynthesis. In the only study on the subject, Annous and Song (9) reported that although pantothenate is phosphorylated within rat erythrocytes (the first step in CoA biosynthesis), there is no evidence for the subsequent steps of the CoA biosynthesis pathway. Saliba et al. (10) confirmed that human erythrocytes similarly phosphorylate pantothenate, but did not investigate whether CoA synthesis also occurs in the cells. A lack of CoA biosynthesis in mammalian erythrocytes would seemingly place the burden of CoA synthesis squarely on malaria parasites that infect mammals (such as P. falciparum), contrary to the situation in birds. Although Saliba et al. (10) have shown that P. falciparum is capable of performing the first step in CoA biosynthesis, it remains to be established whether the parasite can metabolize the 4′-phosphopantothenate generated from pantothenate to CoA or, like P. lophurae, relies on CoA synthesized in the host erythrocyte for its normal growth and replication.In this study we followed the metabolism of pantothenate within uninfected human erythrocytes, P. falciparum-infected human erythrocytes, and isolated P. falciparum parasites. We provide evidence that both uninfected erythrocytes (which we show do take up pantothenate, albeit very slowly) and P. falciparum-infected erythrocytes synthesize CoA from pantothenate. CoA biosynthesis is, however, dramatically higher in the P. falciparum-infected cell. Furthermore, we show that P. falciparum parasites synthesize CoA in the absence of the host erythrocyte, and hence, by contrast with avian malaria parasites, the human malaria parasite does not rely on the host erythrocyte for CoA.     

Mefloquine resistance in Plasmodium falciparum

Mefloquine resistance in Plasmodium falciparum, the most dangerous of the four pathogenic malaria parasites of humans, is established in several endemic regions of the world. After a promising start, resistance has developed to disturbing extents in some areas, whereas in many regions it remains an effective drug. In this article, Frank Mockenhaupt reviews the factors that are likely to influence the development of mefloquine resistance, its possible mechanism and its geographical spread.     

Gametocytemia in Plasmodium vivax and Plasmodium falciparum infections

Two expert research microscopists, each blinded to the other's reports, diagnosed single-species malaria infections in 2,141 adults presenting at outpatient malaria clinics in Tak Province, Thailand, and Iquitos, Peru, in May-August 1998, May-July 1999, and May-June 2001. Plasmodium vivax patients with gametocytemia had higher fever and higher parasitemia than those without gametocytemia; temperature correlated with parasitemia in the patients with gametocytemia. Plasmodium falciparum patients with gametocytemia had lower fever than those without gametocytemia, but similar parasitemia; temperature correlated with parasitemia in the patients without gametocytemia. Hematologic data in Thailand in 2001 showed lower platelet counts in P. vivax patients with gametocytemia than in the P. vivax patients without gametocytemia, whereas P. falciparum patients with gametocytemia had similar platelet counts but lower red blood cell counts, hemoglobin levels, hematocrit levels, and higher lymphocyte counts than patients without gametocytemia.     

Protein dolichylation in Plasmodium falciparum

We performed reverse-phase thin-layer chromatography and reverse-phase high-performance liquid chromatography (RP-HPLC) analysis of polyisoprenoids released by sulfonium-salt cleavage with methyl iodide from Plasmodium falciparum proteins labeled with [3H]FPP or [3H]GGPP and showed that a dolichol of 11 isoprene units is bound to 21-28-kDa protein clusters from trophozoite and schizont stages. The dolichol structure was confirmed by electrospray-ionization mass spectrometry analysis. Treatment with protein synthesis inhibitors and RP-HPLC analysis of the proteolytic digestion products from parasite proteins labeled with [35S]cysteine and [3H]FPP showed that the attachment of dolichol to protein is a post-translational event and probably occurs via a covalent bond to cysteine residues.     

Advances in the Plant Isoprenoid Biosynthesis Pathway and Its Metabolic Engineering

Although the cytosolic isoprenoid biosynthetic pathway, mavolonate pathway, in plants has been known for many years, a new plastidial 1-deoxyxylulose-5-phosphate (DXP) pathway was identified in the past few years and its related intermediates, enzymes, and genes have been characterized quite recently. With a deep insight into the biosynthetic pathway of isoprenoids, investigations into the metabolic engineering of isoprenoid biosynthesis have started to prosper. In the present article, recent advances in the discoveries and regulatory roles of new genes and enzymes in the plastidial isoprenoid biosynthesis pathway are reviewed and examples of the metabolic engineering of cytosolic and plastidial isoprenoids biosynthesis are discussed.     

Plasmodium falciparum glutaredoxin-like proteins

Glutaredoxin-like proteins form a new subgroup of glutaredoxins with a serine replacing the second cysteine in the CxxC-motif of the active site. Yeast Grx5 is the only glutaredoxin-like protein studied biochemically so far. We identified and cloned three genes encoding glutaredoxin-like proteins from the malaria parasite Plasmodium falciparum (Pf Glp1, Pf Glp2, and Pf Glp3) containing a conserved cysteine in the CGFS-, CKFS-, and CKYS-motif, respectively. Here, we describe biochemical properties of Pf Glp1 and Pf Glp2. Cys 99, the only cysteine residue in Pf Glp1, has a pK(a) value as low as 5.5 and is able to mediate covalent homodimerization. Monomeric and dimeric Pf Glp1 react with GSSG and GSH, respectively. Pf Glp2 is monomeric and both of its cysteine residues can be glutathionylated. Molecular models reveal a thioredoxin fold for the putative C-terminal domain of Pf Glp1, Pf Glp2, and Pf Glp3, as well as conserved residues presumably required for glutathione binding. However, Pf Glp1 and Pf Glp2 neither possess activity in a classical glutaredoxin assay nor display activity as glutathione peroxidase or glutathione S-transferase. Mutation of Ser 102 in the CGFS-motif of Pf Glp1 to cysteine did not generate glutaredoxin activity either. We conclude that, despite their ability to react with glutathione, glutaredoxin-like proteins are a mechanistically and functionally heterogeneous group with only little similarities to canonical glutaredoxins.     

Glycobiology of Plasmodium falciparum

The human malaria parasite, Plasmodium falciparum, has as its only glycoconjugate GPI anchors. These structures, present in essentially all parasite surface proteins, are associated with disease pathology. In contrast, the parasite depends for essential recognition events on saccharides associated with host cell glycoproteins and proteoglycans.     

Advances in the Plant Isoprenoid Biosynthesis Pathway and Its Metabolic Engineering

Although the cytosolic isoprenoid biosynthetic pathway, mavolonate pathway, in plants has been known for many years, a new plastidial 1-deoxyxylulose-5-phosphate (DXP) pathway was identified in the past few years and its related intermediates, enzymes, and genes have been characterized quite recently.With a deep insight into the biosynthetic pathway of isoprenoids, investigations into the metabolic engineering of isoprenoid biosynthesis have started to prosper. In the present article, recent advances in the discoveries and regulatory roles of new genes and enzymes in the plastidial isoprenoid biosynthesis path way are reviewed and examples of the metabolic engineering of cytosolic and plastidial isoprenoids biosnthesis are discussed.     

Small GTP-binding proteins in Plasmodium falciparum

Summary— During its erythrocytic life cycle Plasmodium falciparum exchanges compounds with host cells through phagocytosis and exocytosis. In eucaryotic cells, small GTP-binding proteins of the Ras superfamily appear to be involved in different steps of membrane trafficking and in intracellular signals. In this paper, we investigate the Rab4, Rab6 and Ras-related proteins in P falciparum infected red cells. We report that P falciparum Rab and Ras-related proteins could be distinguished from their counterparts by iso-electrofocusing and immunoblotting. The localization of P falciparum Rab 4 and Rab 6 was studied by immunogold electron microscopy on ultrathin frozen sections of infected red blood cells. Rab4 parasite-relate protein was found associated with the membranes of early endosome-like structures near the parasite plasma membrane. Rab6-related protein was associated with the Golgi/trans Golgi network, as already suggested by immunofluorescence microscopy studies and Ras-related protein was cytoplasmic and plasma membrane-associated. These results are in accordance with their mammalian counterparts and support the implication of Rab-related proteins in vesicular trafficking in Plasmodium.