Phosphoenolpyruvate carboxylase dentified as a key enzyme in erythrocytic Plasmodium falciparum carbon metabolism

Phospoenolpyruvate carboxylase (PEPC) is absent from humans but encoded in thePlasmodium falciparum genome, suggesting that PEPC has a parasite-specific function. To investigate its importance in P. falciparum, we generated a pepc null mutant (D10Δpepc), which was only achievable when malate, a reduction product of oxaloacetate, was added to the growth medium. D10Δpepc had a severe growth defect in vitro, which was partially reversed by addition of malate or fumarate, suggesting that pepc may be essential in vivo. Targeted metabolomics using 13C-U-D-glucose and 13C-bicarbonate showed that the conversion of glycolytically-derived PEP into malate, fumarate, aspartate and citrate was abolished in D10Δpepc and that pentose phosphate pathway metabolites and glycerol 3-phosphate were present at increased levels. In contrast, metabolism of the carbon skeleton of 13C,15N-U-glutamine was similar in both parasite lines, although the flux was lower in D10Δpepc; it also confirmed the operation of a complete forward TCA cycle in the wild type parasite. Overall, these data confirm the CO2 fixing activity of PEPC and suggest that it provides metabolites essential for TCA cycle anaplerosis and the maintenance of cytosolic and mitochondrial redox balance. Moreover, these findings imply that PEPC may be an exploitable target for future drug discovery

Use of metabolomics to decipher parasite carbon metabolism of asexual erythrocytic stages of the human malaria parasite Plasmodium falciparum

Le paludisme est une des maladies tropicales les plus dévastatrices au monde causée par des parasites protozoaires intracellulaires du genre Plasmodium. Cinq espèces de plasmodies sont responsables du paludisme chez l'homme et causent 600 000 décès par an principalement chez les enfants de moins de 5 ans et les femmes enceintes vivant dans les régions les plus pauvres du globe. Les parasites ont généré une résistance contre les chimiothérapies existantes et aucun vaccin efficace n'est encore disponible. Il est donc impératif d'identifier et de valider de nouvelles cibles qui peuvent être exploitées pour la découverte de nouveaux médicaments.Cette étude a porté sur la caractérisation d'un enzyme, la phosphoénolpyruvate carboxylase (PEPC), produit d'un gène spécifique au parasite et absent chez l'hôte humain, ce qui constitue l'un des pré-requis d'une cible potentielle pour la découverte de médicaments. Le gène avait été montré comme essentiel pour des parasites seulement en absence de malate ou de fumarate, suggérant un rôle de la protéine dans le métabolisme du carbone intermédiaire des parasites.Mes études de thèse avaient pour but de caractériser le rôle de la PEPC en utilisant la métabolomique. J'ai d'abord établi et normalisé une méthodologie d'analyses métabolomiques des globules rouges infectés par Plasmodium et optimisé l'analyse des métabolites hydrophiles présents dans le parasite intracellulaire et sa cellule hôte. Nous nous sommes concentrés sur les métabolites du métabolisme du carbone intermédiaire, où la PEPC pouvait jouer un rôle déterminant par analogie avec les plantes et les bactéries. Des analyses ciblées utilisant un marquage isotopique du métabolome à partir de 13C-U-glucose, 13C-bicarbonate et 13C, 15N-glutamine ont aussi été réalisées permettant de mieux appréhender les conséquences d'un KO de l'enzyme PEPC sur le métabolisme du parasite.Les données montrent que l'enzyme PEPC permet une fixation du bicarbonate et catalyse une réaction anaplérotique conduisant à du malate qui est introduit dans le cycle de l'acide tricarboxylique mitochondrial, transférant ainsi des équivalents réducteurs du cytoplasme à la mitochondrie et fournissant aussi un point d'entrée du squelette carboné dans le cycle. Les résultats montrent surtout que les parasites possèdent un cycle complet et de type oxydatif de l'acide tricarboxylique mitochondrial. Il parait y avoir trois points d'entrée: 1. l'acétyl CoA résultant du pyruvate généré par la glycolyse et décarboxylé dans la mitochondrie; 2. l'acide alpha-cétoglutarique provenant du glutamate, qui lui-même résulte de la désamination de la glutamine essentiellement fournie par l'environnement externe; 3. le malate, produit en aval de la malate déshydrogénase qui réduit l'oxaloacétate produit par la PEPC. En aval de la PEPC, la biosynthèse des pyrimidines opère grâce à l'activité de l'aspartate aminotransférase agissant sur oxaloacétate.En dehors du malate, le fumarate est le seul autre métabolite qui permet de s'opposer au défaut de croissance des parasites déficients en PEPC, ce qui a conduit à évaluer le rôle de la fumarase. À cette fin, l'étiquetage du gène endogène fumarase avec une étiquette HA, a permis de montrer que la protéine est exprimée dans les stades intra-érythrocytaires de P. falciparum et de montrer que la protéine se trouve à la fois dans la mitochondrie et le cytoplasme. La protéine recombinante a été exprimée avec succès et partiellement caractérisée biochimiquement. De nombreuses tentatives visant à générer des mutants de délétion génétique de P. falciparum n'ont pas abouti, laissant en suspens la question du caractère essentiel du gène pour les parasites. Cependant, il est possible de cibler le locus du gène via un marquage C-terminal. Ceci suggère que l'enzyme peut être essentielle pour la survie du parasite et donc une cible exploitable pour la découverte d'un type nouveau de médicament antipaludique.Malaria is one of the world's most devastating tropical diseases caused by obligate intracellular protozoan parasites of the genus Plasmodium. Five species of these parasites cause malaria in humans and infection results in ~600,000 deaths annually primarily in children under the age of 5 and pregnant women living in the poorest areas of the globe. The parasites have an outstanding ability to generate resistance against existing chemotherapies and an efficacious vaccine is not available yet. Therefore it is imperative that attempts are being made to identify and validate new targets that can be exploited for future drug discovery.This study focused on the validation and elucidation of a parasite-specific gene product namely phosphoenolpyruvate carboxylase (PEPC), which is not present in the human host and thus has one of the pre-requisites of a potential drug target. The gene had been previously genetically validated and it was demonstrated that mutant parasites lacking pepc were only viable in the presence of malate or fumarate, suggesting a role of the protein in intermediary carbon metabolism of the parasites.My studies had the goal to assess the role of PEPC using a metabolomics approach. Initially the methodologies to perform metabolomics analyses of Plasmodium-infected RBCs were established and standardised and it was assessed how to best analyse the hydrophilic metabolites present in the intracellular parasites and its host cell. We focused on metabolites of intermediary carbon metabolism, as it is likely that PEPC is important for metabolic functions linked to this in the parasites, in analogy to plants and bacteria. While global metabolomics analyses were appealing, it was decided to apply a targeted metabolomics and comparative approach using stable isotope labelling of the parasite metabolomes with 13C-U-glucose, 13C-bicarbonate and 13C-,15N-glutamine to assess the consequences of the pepc knockout on parasite metabolism.The data demonstrated that PEPC has an anaplerotic function fixing bicarbonate and leading to generation of malate that is fed into the mitochondrial tricarboxylic acid cycle and so transfers reducing equivalents from cytoplasm to mitochondrion as well as providing an entry point of carbon skeleton into the cycle. The most important findings with respect to parasite mitochondrial metabolism were that the parasites possess a complete and oxidative tricarboxylic acid cycle, which appears to have three entry points: 1. Acetyl CoA resulting from glycolytically generated pyruvate that is decarboxylated in the mitochondrion; 2. α-ketoglutarate from the reaction of glutamate dehydrogenase and 3. malate, which is a downstream product of malate dehydrogenase that reduces oxaloacetate the reaction product of PEPC. Other downstream reactions supported by PEPC activity are pyrimidine biosynthesis through the activity of aspartate aminotransferase also acting on the PEPC-derived oxaloacetate.Apart from malate, fumarate was the only other metabolite that reversed the growth defect of pepc mutant parasites. Hence the role of fumarase in the parasites was also assessed. To this end the endogenous fumarase gene of P. falciparum was tagged with an HA-tag, which showed that the protein is expressed in the intra-erythrocytic stages of P. falciparum and demonstrated that the protein is located in both mitochondrion and cytoplasm. In addition, the recombinant protein was produced and partially biochemically characterised. Numerous independent attempts to generate genetic deletion mutants of P. falciparum were unsuccessful, leaving the question whether the gene is essential for the parasites unanswered. However, it was possible to manipulate the locus by C-terminal tagging of the fumarase gene suggesting that fumarase might be indeed essential for parasite survival and therefore possibly suitable for future drug design and discovery

Location determination Algorithms for Distributed

Wireless ad-hoc networks are self-organizing, rapidly deployable and require no fixed infrastructure. Localization is defined as the ability of the sensors in a network to determine their position in the same co-ordinate system. The most prevalent technique of localization in sensor networks is use of a global positioning system (GPS).However it has the disadvantage of working only outdoors, on lands with sparse foliage environments and generally has high power consumption. Our project aims location determination or localization in Wireless Ad-Hoc sensor networks. It requires a simple average hop distance based algorithm allowing a distributed set of "dumb" nodes to determine their location by calculating their distance to known landmarks, i.e. the "smart nodes". We propose the "Distance Vector -- Hop Propagation" algorithm for localization in sensor networks

Hypothetical model of the contributions PEPC and adaptations of D10^Δ^<i>pepc</i> to compensate for lack of PEPC.

<p>The width of arrows is indicative of the fluxes through the pathways. Glucose used by wild type parasites is phosphorylated into glucose 6-phosphate, which is either catabolised in the glycolytic pathway to generate lactate or is used in the PPP to provide NADPH and 5 and 7-carbon sugars for downstream metabolic processes. During glycolysis, NAD⁺ reduction and NADH oxidation (blue boxes) are balanced in the complete glycolytic sequence itself, but some metabolites are used in other ways. Glycerol 3-phosphate dehydrogenase and especially MDH, converting OAA (the product of CO₂ fixation by PEPC) to malate, play roles in maintenance of redox balance in the cytoplasm (red boxes). Pyruvate is metabolised not only into lactate but is also transferred into the mitochondrion where it is oxidised into acetyl-CoA by a PDH-like dehydrogenase <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003876#ppat.1003876-MacRae1" target="_blank">[3]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003876#ppat.1003876-Cobbold1" target="_blank">[6]</a>. Malate is transferred into the mitochondrion via a malate∶α-ketoglutarate (αKG) antiporter and enters TCA metabolism leading to OAA formation in the organelle, this oxidation transfers reducing equivalents into the mtETC (QH₂). OAA may leave the mitochondrion, thus completing a parasite-specific form of a malate shuttle, or be converted to citrate as part of canonical TCA metabolism. Deletion of <i>pepc</i> leads to an imbalance in redox metabolism in both cytosol and presumably also mitochondrion. D10^{Δ<i>pepc</i>} apparently adapted to this with increased generation of glycerol 3-phosphate with concomitant NADH oxidation, thus compensating partially for the loss of downstream generation of NAD⁺ through MDH. Increased flux through oxidative PPP is a mechanism for generating NADPH which may be compensating for the reduced flux through TCA metabolism and therefore a reduced generation of NADPH at the IDH step in D10^{Δ<i>pepc</i>}(purple boxes). The reduction in flux to lactate in D10^{Δ<i>pepc</i>} reflects the severely reduced growth phenotype of D10^{Δ<i>pepc</i>}. The flux from glutamine via glutamate to α-ketoglutarate and, within the mitochondrion, to succinate and, at an apparently lower flux, to fumarate and malate is shown, with α-ketoglutarate as an additional entry point into TCA metabolism. The flux through these metabolites is lower in D10^{Δ<i>pepc</i>} compared with D10, reflecting the general growth defect of D10^{Δ<i>pepc</i>} and also its reduced consumption of glutamine, although the pathway in which the carbon skeleton is incorporated is not dramatically affected.</p

Metabolite concentrations in spent medium.

<p>All data are means of three independent experiments with standard deviations.</p><p>: Glucose utilisation was significantly lower in D10^{Δ<i>pepc</i>} compared to D10 (p<0.05).</p><p>: Lactate excretion and glutamine utilisation were significantly lower in D10^{Δ<i>pepc</i>} compared to D10 (p<0.001). Succinate, malate and fumarate concentrations in the spent medium were below the detection limit of the assay kits used.</p

Gene replacement of <i>pepc</i>.

<p>(A) Schematic representation of the endogenous <i>pepc</i> locus, the pCC4-Δ<i>pepc</i> plasmid and the <i>pepc</i> locus following integration by double crossover recombination of pCC4-Δ<i>pepc</i> (D10^{Δ<i>pepc</i>}). SpeI restriction sites for diagnostic Southern blot analyses and sizes of the expected DNA fragments are indicated. Two regions homologous to the 5′ (Δ<i>pepc</i> 5′) and 3′ (Δ<i>pepc</i> 3′) end of <i>pepc</i> present in pCC4-Δ<i>pepc</i> recombine with the endogenous <i>pepc</i> locus and part of the <i>pepc</i> gene is replaced with the positive selectable marker <i>blasticidin-S-deaminase</i> (<i>bsd</i>), which is under control of the <i>calmodulin</i> promoter (<i>cam</i> 5′) and the <i>histine rich protein 2</i> 3′ UTR (<i>hrp2</i> 3′). The plasmid contains the negative selectable marker <i>cytosine deaminase</i> (<i>CD</i>), which is under control of the <i>heat shock protein 86</i> promotor (<i>hsp86</i> 5′) and the <i>P. berghei dihydrofolate reductase</i> 3′ UTR (<i>PbDT</i> 3′), and is lost upon double crossover recombination. (B) Southern blot of SpeI-digested genomic DNA of wild type parasites and parasites transfected with pCC4-Δ<i>pepc</i>, cultured in routine medium or malate medium, probed with the 5′ end of <i>pepc</i>. Cycle 1 (c1) refers to the first parasites resistant to blasticidin and 5-fluorocytosine (5-FC), while cycle 2 (c2) are 5-FC-resistant parasites after 1 additional drug selection cycle. Integration of the plasmid only occurred when the transfectants were cultured in malate medium (diagnostic fragment: 5.0 kb). The derived clones D10^{Δ<i>pepc</i>}-1 and D10^{Δ<i>pepc</i>}-2 are shown in lane 5 and 6. In routine medium no integration was observed, only fragments corresponding to endogenous <i>pepc</i> (7.1 kb), plasmid (6.2 kb and 2.1 kb) and five fragments of unknown identity (*) were detected.</p

Efficacy of L-cycloserine, DSM190 and atovaquone against D10 and the D10^Δ^<i>pepc</i> in routine and malate media.

<p>The effect of L-cycloserine (A), DSM190 (B) and atovaquone (C) on parasite viability. D10^{Δ<i>pepc</i>} was maintained for 9 days in routine medium prior to the experiment.</p

Metabolite labelling from ¹³C-bicarbonate.

<p>(A) Schematic representing bicarbonate utilisation in <i>P. falciparum</i> by determining the incorporation of the single ¹³C isotope into metabolic intermediates. (B) ¹³C-bicarbonate is incorporated into malate and fumarate and also to a lesser extent into aspartate and citrate. The natural relative abundance of ¹³C-1 label in metabolites was not subtracted from the data shown. This accounts for ∼1.1% of ¹³C-1 for each carbon of the metabolites in all samples analysed including those of D10^{Δ<i>pepc</i>} (Δ<i>pepc</i>) and RBC, which show no specific additional incorporation of ¹³C-bicarbonate into their carbon skeleton. Thus the specific incorporation into the metabolites is most usefully assessed by comparing the levels in parasite-infected cells with that measured in the RBC samples (which reflects the natural abundance of the isotope). Abbreviations: as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003876#ppat-1003876-g005" target="_blank">Figure 5</a> and m, spent medium samples.</p

Growth phenotype of D10^Δ^<i>pepc</i> and rescue by malate and fumarate.

<p>(A) Growth of D10 in routine medium lacking malate and D10^{Δ<i>pepc</i>} in malate or routine medium was followed for 14 days. The cultures were diluted 1∶5 on even days. The means ± S.D. of two (D10) and three (D10^{Δ<i>pepc</i>}) replicates are shown. (B and C) D10 and D10^{Δ<i>pepc</i>} were cultured in routine medium for 9 days, synchronised, diluted to 1% parasitaemia and cultured for 5 days in medium supplemented with increasing concentrations of malate (0.5–5 mM) (B) or a range of metabolites (C). The cultures were diluted 1∶5 on day 3. The parasitaemia on day 5 was determined and multiplied with the dilution factor to give the accumulated parasitaemia. Data are means ± S.E.M. of at least 3 independent experiments, each done in triplicate (for succinate and citrate, means ± S.D. of 2 experiments done in triplicate). The asterisks indicate a statistically significant increase in parasitaemia compared to routine medium (p<0.05). Other apparent changes were not statistically significant. The final concentrations of metabolites used in (C) were: 0.5 mM citrate, 5 mM all others.</p

Metabolite labelling from ¹³C-U-D-glucose.

<p>(A) Schematic representing glucose utilisation in <i>P. falciparum</i> based on the utilisation of ¹³C-U-D-glucose and distribution of ¹³C carbons into metabolic intermediates. (B) The parasites perform glycolysis such that triose 3-phosphates, phospho<i>enol</i>pyruvate (PEP) and lactate had very extensive labelling with ¹³C from ¹³C-U-D-glucose (all are three carbon compounds, hence exist mainly as ¹³C-3 molecules as rapidly generated from glucose by glycolysis). ¹³C is also incorporated into malate, fumarate and aspartate through the action of PEPC (and hence the ¹³C-3 molecules) as well as the TCA metabolite citrate by the action of a PDH-like enzyme <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003876#ppat.1003876-MacRae1" target="_blank">[3]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003876#ppat.1003876-Cobbold1" target="_blank">[6]</a> in D10 parasites. Citrate is formed from OAA (occurring as ¹³C-3 molecule as well as unlabelled) and acetyl-CoA (presumably mainly ¹³C-2 molecules as generated from pyruvate) and so was present as ¹³C-2 and ¹³C-5 molecules as well as unlabelled. ¹³C-labelled malate, fumarate, asparate and citrate were absent from samples of D10^{Δ<i>pepc</i>} (Δ<i>pepc</i>). ¹³C-U-D-glucose was also fed into the PPP intermediates ribulose 5-P/ribose 5-P and sedoheptulose 7-P (compounds with 5 and 7 carbons, respectively, hence the extensive presence as ¹³C-5 and ¹³C-7 molecules) and used to generate glycerol 3-phosphate (also a 3-carbon molecule hence the ¹³C-3 labelling). Legend: UL, unlabelled; ¹³C-1: one carbon atom labelled; ¹³C-2 to ¹³C-7: two to seven carbon atoms labelled; D10⁹⁰, D10-infected RBC concentrated to 90% parasitaemia; D10⁶, D10-infected RBC at 6% parasitaemia. (C) Relative levels of intracellular metabolites in D10^{Δ<i>pepc</i>} (Δ<i>pepc</i>) and D10. Abbreviations: α-KG, α-ketoglutarate; DHAP, dihydroxyacetone phosphate; GAP, glyceraldehyde 3-phosphate; OAA, oxaloacetate; PEP, phospho<i>enol</i>pyruvate; PPM, parasite plasma membrane; PVM, parasitophorous vacuole membrane; QH₂, ubiquinol.</p