The membrane potential of the intraerythrocytic malaria parasite Plasmodium falciparum

The membrane potential (Deltapsi) of the mature asexual form of the human malaria parasite, Plasmodium falciparum, isolated from its host erythrocyte using a saponin permeabilization technique, was investigated using both the radiolabeled Deltapsi indicator tetraphenylphosphonium ([(3)H]TPP(+)) and the fluorescent Deltapsi indicator DiBAC(4)(3) (bis-oxonol). For isolated parasites suspended in a high Na(+), low K(+) solution, Deltapsi was estimated from the measured distribution of [(3)H]TPP(+) to be -95 +/- 2 mV. Deltapsi was reduced by the specific V-type H(+) pump inhibitor bafilomycin A(1), by the H(+) ionophore CCCP, and by glucose deprivation. Acidification of the parasite cytosol (induced by the addition of lactate) resulted in a transient hyperpolarization, whereas a cytosolic alkalinization (induced by the addition of NH(4)(+)) resulted in a transient depolarization. A decrease in the extracellular pH resulted in a membrane depolarization, whereas an increase in the extracellular pH resulted in a membrane hyperpolarization. The parasite plasma membrane depolarized in response to an increase in the extracellular K(+) concentration and hyperpolarized in response to a decrease in the extracellular K(+) concentration and to the addition of the K(+) channel blockers Ba(2+) or Cs(+) to the suspending medium. The data are consistent with Deltapsi of the intraerythrocytic P. falciparum trophozoite being due to the electrogenic extrusion of H(+) via the V-type H(+) pump at the parasite surface. The current associated with the efflux of H(+) is countered, in part, by the influx of K(+) via Ba(2+)- and Cs(+)-sensitive K(+) channels in the parasite plasma membrane.     

Inhibition of intraerythrocytic development of Plasmodium falciparum by proteinase inhibitors

A group of inactivators of cysteinyl proteinases which function by covalent bond formation have been examined for their ability to inhibit the development of Plasmodium falciparum within red blood cells. The most effective of these caused inactivation of the parasite near 10(-8) M concentration. The range of inhibitory action varied with peptide structure in a manner characteristic of affinity labels for proteinases suggesting that the target of inhibition was an unidentified proteinase, probably of the cysteinyl type, but different from cathepsins B and L.     

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).     

Glucosamine inhibits inositol acylation of the glycosylphosphatidylinositol anchors in intraerythrocytic Plasmodium falciparum

Glycosylphosphatidylinositol (GPI) anchors are crucial for the survival of the intraerythrocytic stage Plasmodium falciparum because of their role in membrane anchoring of merozoite surface proteins involved in parasite invasion of erythrocytes. Recently, we showed that mannosamine can prevent the growth of P. falciparum by inhibiting the GPI biosynthesis. Here, we investigated the effect of isomeric amino sugars glucosamine, galactosamine, and their N-acetyl derivatives on parasite growth and GPI biosynthesis. Glucosamine, but not galactosamine, N-acetylglucosamine, and N-acetylgalactosamine inhibited the growth of the parasite in a dose-dependent manner. Glucosamine specifically arrested the maturation of trophozoites, a stage at which the parasite synthesizes all of its GPI anchor pool and had no effect during the parasite growth from rings to early trophozoites and from late trophozoites to schizonts and merozoites. An analysis of GPI intermediates formed when parasites incubated with glucosamine indicated that the sugar interferes with the inositol acylation of glucosamine-phosphatidylinositol (GlcN-PI) to form GlcN-(acyl)PI. Consistent with the non-inhibitory effect on parasite growth, galactosamine, N-acetylglucosamine, and N-acetylgalactosamine had no significant effect on the parasite GPI biosynthesis. The results indicate that the enzyme that transfers the fatty acyl moiety to inositol residue of GlcN-PI discriminates the configuration at C-4 of hexosamines. An analysis of GPIs formed in a cell-free system in the presence and absence of glucosamine suggests that the effect of the sugar is because of direct inhibition of the enzyme activity and not gene repression. Because the fatty acid acylation of inositol is an obligatory step for the addition of the first mannosyl residue during the biosynthesis of GPIs, our results offer a strategy for the development of novel anti-malarial drugs. Furthermore, this is the first study to report the specific inhibition of GPI inositol acylation by glucosamine in eukaryotes.     

Evidence that Plasmodium falciparum diacylglycerol acyltransferase is essential for intraerythrocytic proliferation

In triacylglycerol (TAG)-accumulating organisms, the physiological roles of diacylglycerol acyltransferase (DGAT), a principal enzyme in the major biosynthetic pathway for TAG, appear to be diverse. Apicomplexan parasite, Plasmodium falciparum, shows unique features in TAG metabolism and trafficking during intraerythrocytic development, and unlike most eukaryotes, only one open reading frame (ORF) encoding a candidate DGAT could be found in its genome. However, whether this candidate ORF encodes P. falciparum DGAT and its physiological relevance have not been assessed. Here, we demonstrate that the ORF is transcribed as a approximately 3.6 kb single mRNA throughout intraerythrocytic development, markedly elevated at trophozoite, schizont, and segmented schizont, and indeed encodes a protein exhibiting DGAT activity. Further, we provide evidence that the parasite in which the ORF was disrupted via double crossover recombination cannot be enriched, implying a fundamental role of PfDGAT in intraerythrocytic proliferation.     

Apparent lack of N-glycosylation in the asexual intraerythrocytic stage of Plasmodium falciparum.

This study investigates protein glycosylation in the asexual intraerythrocytic stage of the malaria parasite, Plasmodium falciparum, and the presence in the infected erythrocyte of the respective precursors. In in vitro cultures, P. falciparum can be metabolically labeled with radioactive sugars, and its multiplication can be affected by glycosylation inhibitors, suggesting the capability of the parasite to perform protein-glycosylation reactions. Gel-filtration analysis of sugar-labeled malarial proteins before and after specific cleavage of N-glycans or O-glycans, respectively, revealed the majority of the protein-bound sugar label to be incorporated into O-glycans, but only little (7-12% of the glucosamine label) or no N-glycans were found. Analysis of the nucleotide sugar and sugar-phosphate fraction showed that radioactive galactose, glucosamine, fucose and ethanolamine were converted to their activated derivatives required for incorporation into protein. Mannose was mainly recovered as a bisphosphate, whereas the level of radiolabeled GDP-mannose was below the detection limit. The analysis of organic-solvent extracts of sugar-labeled cultures showed no evidence for the formation by the parasite of dolichol cycle intermediates, the dedicated precursors in protein N-glycosylation. Consistently, the amount of UDP-N-acetylglucosamine formed did not seem to be affected by the presence of tunicamycin in the culture. Oligosaccharyl-transferase activity was not detectable in a lysate of P. falciparum, using exogenous glycosyl donors and acceptors. Our studies show that O-glycosylation is the major form of protein glycosylation in intraerythrocytic P. falciparum, whereas there is little or no protein N-glycosylation. A part of these studies has been published in abstract form [Dieckmann-Schuppert, A., Hensel, J. and Schwarz, R. T. (1991) Biol. Chem. Hoppe-Seyler 372, 645].     

Effects of mitochondrial inhibitors on intraerythrocytic Plasmodium falciparum in in vitro cultures

Malarial parasites infecting mammalian hosts are considered to be homolactate fermentors at their asexual intraerythrocytic developmental stage; however, existing ultrastructural and biochemical evidence suggest that their acristate mitochondria could be involved in energy metabolism. In the present study, inhibitors of mitochondrial function including compounds which act on NADH and succinate dehydrogenases, electron transport and mitochondrial ATPase, as well as uncouplers, were found to inhibit the growth and propagation of the human parasite Plasmodium falciparum in in vitro cultures at concentrations that specifically affect mitochondrial functions. Direct measurement of parasite protein and nucleic acid synthesis in synchronized cultures showed that throughout the parasite life cycle both processes were inhibited, the latter process being more sensitive. These results strongly suggest that intraerythrocytic malarial parasites require mitochondrial energy production.     

Requirements for maximal enrichment of viable intraerythrocytic Plasmodium falciparum rings by saponin hemolysis

The purpose of the present study was to confirm the effectiveness of saponin hemolysis for concentrating ring-infected erythrocytes in Plasmodium falciparum cultures and to determine the actual numbers of the enriched parasites, not just percentage parasitemia. This is important because various molecular biology and vaccine development against malaria require useable quantities of pure culture with minimal number of uninfected erythrocytes at all stages. Synchronized cultures of three P. falciparum strains were exposed to 0.015% isotonic saponin solution for 30 minutes on ice. They were centrifuged and the pellets were treated again with saponin solution for 3-7 minutes. Initially, most of the cultures contained approximately 10(10) erythrocytes and 1-7% parasitemia, but at the end of the enrichment up to 10(8) of erythrocytes containing 90-99.8% parasitemia were recovered (maximal enrichment). From microscopic examination of the cells it was calculated that the hemolysis rate of uninfected and infected erythrocytes was circa 27 to 1, which could account for the enrichment. Studies by other investigators have suggested that P. falciparum merozoite invasion decreases erythrocyte membrane lipids, and it has been reported that reduction of membrane cholesterol could make erythrocytes saponin-resistant. The possibility that merozoite invasion made erythrocytes partially resistant to saponin hemolysis was strengthened by the observation that the proportions of multiple infections increased significantly in the enriched cultures. However, mature asexual parasites could not be concentrated by this method, suggesting possible differences between the membranes of erythrocytes containing ring forms and those of trophozoites and schizonts. Ring-infected erythrocytes freshly from malaria patients could also not be concentrated by the method described here, suggesting that the ability to induce saponin resistance in erythrocytes was acquired by the parasites in vitro.     

Pyrimidine de novo synthesis during the life cycle of the intraerythrocytic stage of Plasmodium falciparum

The 6 enzymes involved in de novo synthesis of pyrimidines were measured in Plasmodium falciparum isolated by saponin lysis from RBC's nonsynchronized and synchronized in vitro cultures. The total activities were found to be dependent on the stage of the P. falciparum cycle. In parasites isolated from synchronized cultures, the highest activities for all enzymes were found at about 27 hr after synchronization in the late trophozoite stage, or just before schizont formation. Merozoites and ring forms contained little de novo activity. The first enzyme of the pathway, carbamyl phosphate synthetase (CPS-II) preferentially utilized glutamine. Ammonia was a poor substrate. CPS-II was unstable in the absence of the cryoprotectants, dimethylsulfoxide and glycerol. The apparent Km for MgATP--was 3.8 +/- 0.7 mM and the enzyme in all morphological forms of P. falciparum (ring, mature trophozoites and schizonts) was inhibited by UTP. The activity of the fourth enzyme of the pathway, dihydroorotate dehydrogenase, appeared to be linked to the cell's respiratory chain; inhibitors of mammalian electron transport such as cyanide, amytal, antimycin A, thenoyltrifluoroacetone and ubiquinone analogs also inhibited the P. falciparum enzyme. The demonstration of the variation of activity of the pyrimidine enzymes correlates with the increased synthesis of nucleic acids in the late trophozoite stage. These observations provide a basis for the testing of the effectiveness of pyrimidine analogs as potential antimetabolites against various forms of the parasite.     

Effects of chloroquine on the feeding mechanism of the intraerythrocytic human malarial parasite Plasmodium falciparum

Ultrastructural investigations of P. falciparum cultivated in vitro in human erythrocytes revealed new features of the feeding mechanism of the parasite. Mature trophozoites and schizonts take up a portion of the host cytosol by endocytosis which is restricted to cytostomes and which involves the invagination of both parasitophorous and parasite membranes. The resulting endocytic vesicles, surrounded by two concentric membranes, migrate towards the central food vacuole membrane. The external membrane of the endocytic vesicles apposes that of the food vacuole, leading to the internalization of vesicles bounded by a single membrane into the vacuole space where they are rapidly degraded. We conclude from this sequence of events that endocytic vesicles fuse with the food vacuole. Treatment of infected cells with therapeutic concentrations of chloroquine inhibited the last step of the feeding process, i.e. vacuolar degradation. This was manifested by the accumulation within the vacuolar space of intact vesicles bounded by single membranes. The implications of these findings for the antimalarial activity of chloroquine are discussed.     

Serum factors governing intraerythrocytic development and cell cycle progression of Plasmodium falciparum.

Malaria is clinically manifested only when the human malaria parasites in the genus Plasmodium enter the obligatory intraerythrocytic life cycle. Elucidation for the roles of the serum, the key nutrient, and its components is then deemed essential for thorough understanding of the proliferation of Plasmodium cells at the erythrocytic stage. Fractionation and analysis of serum and its components was performed by chromatography, solvent extraction, and subsequent reconstitution experiments. Only fractions containing serum albumin (SA) from the serum and purified intact bovine serum albumin (BSA) showed comparable growth promoting activity with human serum (HS). Delipidated BSA can only effect parasite growth after reconstitution with lipids extracted from intact BSA. Fatty acid (FA) species in the neutral lipid fraction from intact BSA proved likewise when reconstituted with delipidated BSA. Furthermore, the involved FA species have to come in a pair of one saturated and one unsaturated, with palmitic and oleic acids as the best combination. The results were further substantiated by morphological analysis as well as biochemical analysis of the DNA synthesis during the intraerythrocytic development. This study can be a basis to explore the molecular mechanism of lipid traffic within the parasitized red blood cell (RBC), which can be an important adjunct to the development of drugs for malaria therapy.     

The methylerythritol phosphate pathway is functionally active in all intraerythrocytic stages of Plasmodium falciparum

Two genes encoding the enzymes 1-deoxy-D-xylulose-5-phosphate synthase and 1-deoxy-D-xylulose-5-phosphate reductoisomerase have been recently identified, suggesting that isoprenoid biosynthesis in Plasmodium falciparum depends on the methylerythritol phosphate (MEP) pathway, and that fosmidomycin could inhibit the activity of 1-deoxy-D-xylulose-5-phosphate reductoisomerase. The metabolite 1-deoxy-D-xylulose-5-phosphate is not only an intermediate of the MEP pathway for the biosynthesis of isopentenyl diphosphate but is also involved in the biosynthesis of thiamin (vitamin B1) and pyridoxal (vitamin B6) in plants and many microorganisms. Herein we report the first isolation and characterization of most downstream intermediates of the MEP pathway in the three intraerythrocytic stages of P. falciparum. These include, 1-deoxy-D-xylulose-5-phosphate, 2-C-methyl-D-erythritol-4-phosphate, 4-(cytidine-5-diphospho)-2-C-methyl-D-erythritol, 4-(cytidine-5-diphospho)-2-C-methyl-D-erythritol-2-phosphate, and 2-C-methyl-D-erythritol-2,4-cyclodiphosphate. These intermediates were purified by HPLC and structurally characterized via biochemical and electrospray mass spectrometric analyses. We have also investigated the effect of fosmidomycin on the biosynthesis of each intermediate of this pathway and isoprenoid biosynthesis (dolichols and ubiquinones). For the first time, therefore, it is demonstrated that the MEP pathway is functionally active in all intraerythrocytic forms of P. falciparum, and de novo biosynthesis of pyridoxal in a protozoan is reported. Its absence in the human host makes both pathways very attractive as potential new targets for antimalarial drug development.     

Influence of tumor necrosis factor-alpha on the ability of monocytes and lymphocytes to destroy intraerythrocytic Plasmodium falciparum in vitro

It has been shown that administration of TNF-alpha causes an increase of survival of plasmodium-infected mice. However, this anti-parasitic effect cannot be reproduced in vitro upon direct incubation of the cytokine with the parasite. This suggests that TNF-alpha may act through modulation of some plasmodicidal mechanism not yet clarified. We evaluated the effect of exogenous TNF-alpha on the phagocytosis of Plasmodium falciparum-infected erythrocytes by monocytes and its influence on the ability of monocytes and lymphocytes to inhibit parasite growth. The capacity of endogenous TNF-alpha to influence the ability of monocytes to inhibit the parasite was also verified. We found that addition of 33 ng TNF-alpha/mL to cultures of human monocytes and P. falciparum-infected erythrocytes increased the phagocytic index from 3.8 to 7.8 in the presence of serum containing P. falciparum antibody. TNF-alpha increased the capacity of monocyte plus lymphocyte to inhibit parasite growth by about 3 times at 0.5 and 5 ng/mL. Sera from severely ill P. falciparum-infected individuals inhibited the parasite growth, but addition of anti-TNF-alpha antibody was unable to modify this inhibition. These data show that TNF-alpha can increase the phagocytic capacity. This was probably due to an increased expression of Fc receptors on monocytes or to the modulation of Fc receptor signaling pathways by signals originating from the binding of TNF-alpha to its receptors. TNF-alpha also acted on lymphocytes plus monocytes by increasing the inhibition of P. falciparum by a mechanism not related to phagocytosis. These findings suggest that TNF-alpha has a pleiotropic anti-malaria effect and that this protective effect depends on the interplay of different factors, such as monocytes/macrophages, lymphocytes, and antibodies, in addition to other cells and molecules.     

Plasmodium falciparum: food vacuole localization of nitric oxide-derived species in intraerythrocytic stages of the malaria parasite

Nitric oxide (NO) has diverse biological functions. Numerous studies have documented NO’s biosynthetic pathway in a wide variety of organisms. Little is known, however, about NO production in intraerythrocytic Plasmodium falciparum. Using diaminorhodamine-4-methyl acetoxymethylester (DAR-4M AM), a fluorescent indicator, we obtained direct evidence of NO and NO-derived reactive nitrogen species (RNS) production in intraerythrocytic P. falciparum parasites, as well as in isolated food vacuoles from trophozoite stage parasites. We preliminarily identified two gene sequences that might be implicated in NO synthesis in intraerythrocytic P. falciparum. We showed localization of the protein product of one of these two genes, a molecule that is structurally similar to a plant nitrate reductase, in trophozoite food vacuole membranes. We confirmed previous reports on the antiproliferative effect of NOS (nitric oxide synthase) inhibitors in P. falciparum cultures; however, we did not obtain evidence that NOS inhibitors had the ability to inhibit RNS production or that there is an active NOS in mature forms of the parasite. We concluded that a nitrate reductase activity produce NO and NO-derived RNS in or around the food vacuole in P. falciparum parasites. The food vacuole is a critical parasitic compartment involved in hemoglobin degradation, heme detoxification and a target for antimalarial drug action. Characterization of this relatively unexplored synthetic activity could provide important clues into poorly understood metabolic processes of the malaria parasite.     

Plasmodium falciparum: Chemically defined medium for continuous intraerythrocytic growth using lipids and recombinant albumin

Dioleoylphosphatidylcholine and other phosphatidylcholines containing different fatty acid moieties were found to increase the ability of nonesterified fatty acids (NEFA) to sustain continuous intraerythrocytic growth of Plasmodium falciparum in the presence of specific proteins. Other phospholipids, including phosphatidylethanolamine, phosphatidylserine, and phosphatidic acid, were beneficial to parasite growth. Different combinations and concentrations of NEFA tested in the presence of phospholipids and bovine albumin had variable effects on parasite growth. The most effective combination for promoting parasite growth consisted of 30 μg/ml cis-9-octadecenoic acid (oleic acid) plus 15 μg/ml hexadecanoic acid (palmitic acid). Recombinant human albumin could replace bovine or human albumin in culture media enriched with structurally defined lipids. This study therefore established a chemically defined culture medium suitable for sustaining the growth of P. falciparum.