Metabolite profiling of the intraerythrocytic malaria parasite Plasmodium falciparum by 1 H NMR spectroscopy

In aerobic respiration, the tricarboxylic acid cycle is pivotal to the complete oxidation of carbohydrates, proteins, and lipids to carbon dioxide and water. Plasmodium falciparum, the causative agent of human malaria, lacks a conventional tricarboxylic acid cycle and depends exclusively on glycolysis for ATP production. However, all of the constituent enzymes of the tricarboxylic acid cycle are annotated in the genome of P. Plasmodium falciparum is a parasitic protozoan, which causes the most severe form of malaria in humans. Because of the emergence in the parasite of widespread drug resistance to most of the first-line antimalarials, the development of new drugs that target the parasite metabolism is needed. Over several years, malarial parasites have been studied to understand various novel biochemical features, which not only gives opportunities for chemotherapeutic interventions but also stimulates broader scientific interests.During the intraerythrocytic stages of P. falciparum, the tricarboxylic acid cycle does not seem to function like a conventional tricarboxylic acid cycle for the following reasons. (a) The bulk of the glucose is metabolized to lactate by anaerobic glycolysis, which is then secreted by the parasite as a metabolic waste (1) (2). As a result, unlike aerobic cells, in P. falciparum there is minimal carbon flow from the cytoplasm to the mitochondrial tricarboxylic acid cycle. (b) The multi-enzyme complex pyruvate dehydrogenase, which channels pyruvate into the tricarboxylic acid cycle through acetyl-CoA, has been found to localize on the apicoplast membrane in P. falciparum (3), unlike mammalian cells, where the enzyme is present on the inner mitochondrial membrane (4). (c) The enzyme isocitrate dehydrogenase generates NADPH rather than NADH (5), thereby implying that its main role is probably to act as a redox sensor rather than donating reducing equivalents to the electron transport chain. In addition, P. falciparum synthesizes ATP mainly by substrate level phosphorylation through glycolysis and not through oxidative phosphorylation (6), suggesting that the primary function of the tricarboxylic acid cycle in generating reducing equivalents for the electron transport chain might be dispensable for the parasite. However, genes for all of the enzymes of the tricarboxylic acid cycle are present and are expressed in P. falciparum (7,8) during the intraerythrocytic stages, thereby implying that the pathway probably has important, yet unidentified biosynthetic functions.Unlike its human host, P. falciparum lacks the de novo purine biosynthetic pathway and is hence completely dependent on the purine salvage pathway to meet its purine nucleotide requirements (9). In the purine salvage pathway, hypoxanthine salvaged from the host is phosphoribosylated to IMP, which then branches out to form AMP and GMP (Fig. 1). IMP is converted to AMP in two steps, catalyzed by two distinct enzymes: (a) adenylosuccinate synthetase, which adds a molecule of aspartate to the 6-oxo position of IMP with a concomitan...

Section: Discussionmentioning

confidence: 99%

Metabolic Fate of Fumarate, a Side Product of the Purine Salvage Pathway in the Intraerythrocytic Stages of Plasmodium falciparum

Bulusu¹,

Jayaraman²,

Balaram

2011

“…3B). However, both these metabolites were also significantly increased in the untreated parasites, and GABA was recently reported to be one of the most abundant metabolites in isolated trophozoites (61). Several other relevant metabolites, namely spermine, dcAdoMet, and PLP, were not detected or could not be reliably quantified.…”

Section: Perturbation-specific Compensatory Mechanismsmentioning

confidence: 90%

Co-inhibition of Plasmodium falciparum S-Adenosylmethionine Decarboxylase/Ornithine Decarboxylase Reveals Perturbation-specific Compensatory Mechanisms by Transcriptome, Proteome, and Metabolome Analyses

Brummelen

Olszewski

Wilinski

et al. 2009

Polyamines are ubiquitous components of all living cells, and their depletion usually causes cytostasis, a strategy employed for treatment of West African trypanosomiasis. To evaluate polyamine depletion as an anti-malarial strategy, cytostasis caused by the co-inhibition of S-adenosylmethionine decarboxylase/ ornithine decarboxylase in Plasmodium falciparum was studied with a comprehensive transcriptome, proteome, and metabolome investigation. Highly synchronized cultures were sampled just before and during cytostasis, and a novel zero time point definition was used to enable interpretation of results in lieu of the developmentally regulated control of gene expression in P. falciparum. Transcriptome analysis revealed the occurrence of a generalized transcriptional arrest just prior to the growth arrest due to polyamine depletion. However, the abundance of 538 transcripts was differentially affected and included three perturbation-specific compensatory transcriptional responses as follows: the increased abundance of the transcripts for lysine decarboxylase and ornithine aminotransferase and the decreased abundance of that for S-adenosylmethionine synthetase. Moreover, the latter two compensatory mechanisms were confirmed on both protein and metabolite levels confirming their biological relevance. In contrast with previous reports, the results provide evidence that P. falciparum responds to alleviate the detrimental effects of polyamine depletion via regulation of its transcriptome and subsequently the proteome and metabolome.Polyamines such as putrescine, spermidine, and spermine are small organic compounds containing two or more amino groups. At physiological pH, these polycations interact electrostatically with numerous anionic macromolecules, thereby stabilizing DNA, RNA, nucleoside triphosphates (e.g. ATP), phospholipids, and proteins (1, 2). These interactions with polyamines can alter DNA conformation, regulate replication and transcription, strengthen membranes, regulate ion channels, and protect DNA and phospholipids from oxidative stress (1-5). Yet polyamines are also implicated in apoptosis (5). Polyamine depletion generally causes cytostasis or growth arrest, which implies that these molecules are involved in cell cycle progression and regulation, and it is speculated that polyamines regulate cyclin degradation (1, 6, 7). Therefore, polyamines are essential for cellular growth, differentiation, and macromolecular synthesis and are ubiquitous components of all living cells, except two orders of Archaea (1). Polyamine metabolism is particularly important in rapidly proliferating cells and has been exploited in the treatment of cancer (1) and parasitic diseases (8). Polyamine metabolism of the malaria parasite Plasmodium falciparum is also a potential target for therapeutic intervention (9, 10).Polyamine and methionine metabolism are closely connected. This is particularly evident in Plasmodium where the two rate-limiting enzymes of polyamine biosynthesis, ornithine decarboxylase (ODC) 2 and S-adenosylme...

“…The radiolabeled metabolites detected in extracts prepared as described above were compared with those detected in extracts prepared essentially according to a published methanol/chloroform/water extraction procedure (18). Following extraction, the upper methanol/water phase was collected and dried, then re-dissolved in 200 l of 50 mM Tris-HCl, pH 7.4, at 4°C, and 10 mM dithiothreitol was added.…”

Section: Metabolic Radiolabeling and Extraction Of Radiolabeled Metabmentioning

confidence: 99%

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

Spry

Saliba

2009

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 [ 14 C]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 B 5 ) 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).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 su...