Plasmodium falciparum glutamate dehydrogenase a is dispensable and not a drug target during erythrocytic development

<p>Background: Plasmodium falciparum contains three genes encoding potential glutamate dehydrogenases. The protein encoded by gdha has previously been biochemically and structurally characterized. It was suggested that it is important for the supply of reducing equivalents during intra-erythrocytic development of Plasmodium and, therefore, a suitable drug target.</p> <p>Methods: The gene encoding the NADP(H)-dependent GDHa has been disrupted by reverse genetics in P. falciparum and the effect on the antioxidant and metabolic capacities of the resulting mutant parasites was investigated.</p> <p>Results: No growth defect under low and elevated oxygen tension, no up-or down-regulation of a number of antioxidant and NADP(H)-generating proteins or mRNAs and no increased levels of GSH were detected in the D10(Delta gdha) parasite lines. Further, the fate of the carbon skeleton of [(13)C] labelled glutamine was assessed by metabolomic studies, revealing no differences in the labelling of a-ketoglutarate and other TCA pathway intermediates between wild type and mutant parasites.</p> <p>Conclusions: First, the data support the conclusion that D10(Delta gdha) parasites are not experiencing enhanced oxidative stress and that GDHa function may not be the provision of NADP(H) for reductive reactions. Second, the results imply that the cytosolic, NADP(H)-dependent GDHa protein is not involved in the oxidative deamination of glutamate but that the protein may play a role in ammonia assimilation as has been described for other NADP(H)dependent GDH from plants and fungi. The lack of an obvious phenotype in the absence of GDHa may point to a regulatory role of the protein providing glutamate (as nitrogen storage molecule) in situations where the parasites experience a limiting supply of carbon sources and, therefore, under in vitro conditions the enzyme is unlikely to be of significant importance. The data imply that the protein is not a suitable target for future drug development against intra-erythrocytic parasite development.</p&gt

Glutathione metabolism of Plasmodium falciparum

Apicomplexan parasites of the genus Plasmodium are the causative agent of malaria, one of the most prevalent infectious diseases worldwide. Five different Plasmodium species can cause malaria in humans, leading to a total of approximately 500 million cases each year and of these, P. falciparum causes the most deadly form of the disease and is responsible for more than 1 million deaths annually. A major problem in the global fight against malaria is the widespread resistance of the parasites against the currently available drugs. It is of great importance to identify new drug target as well as to understand the mechanisms that lead to drug resistance in the first instance in order to potentially reverse the resistant phenotypes and to avoid the development of resistance in the future. The tripeptide glutathione (GSH) or γ-glutamylcysteinyl-glycine is the most abundant low molecular weight thiol in most eukaryotic organisms and serves a number of important functions as sulfhydryl-buffer, cofactor for enzymes and for the detoxification of xenobiotics and drugs. GSH is an important component of the antioxidant machinery and because malaria parasites live in an environment rich in iron and oxygen and thus increased oxidative stress, they depend on functional antioxidant systems. The biosynthesis pathway for GSH, consisting of γ-glutamylcysteine synthetase (γGCS) and glutathione synthetase (GS) is present in malaria parasites as well as in their host cells. Previous studies have shown that depletion of GSH has an antimalarial effect, but it remained unclear whether parasites were killed directly or died because their host cell could not survive the depletion of GSH. To address this question, the knockout of both genes encoding the enzymes of the GSH biosynthesis pathway in P. falciparum was attempted. While both gene loci were targeted by control constructs, the knockout of either pfγgcs or pfgs was impossible, indicating both genes are essential for parasite survival in the erythrocytic stages. To analyse the localization of γGCS and GS, GFP-tagged recombinant fusion proteins were expressed in the parasites and showed that GSH biosynthesis is cytosolic. Apart form its other functions GSH has previously been suggested to be involved in resistance to the antimalarial drug chloroquine (CQ). CQ was for a long time the first line antimalarial drug due to its high efficiency, low cost and low toxicity, but is now widely inefficient in the treatment of the disease. CQ resistance is associated with mutations in the CQ resistance transporter (PfCRT), a membrane protein of the digestive vacuole that allows the efflux of the drug form its site of action. However, PfCRT mutations alone cannot explain the full array of phenotypes found in resistant parasites. GSH is able to degrade heme, the target of CQ, in vitro and it has been suggested that elevated GSH levels contribute to CQ resistance. However, analyses of isogenic parasite lines bearing different forms of PfCRT in this study revealed lower GSH levels and higher susceptibility to inhibition of GSH biosynthesis in the CQ resistant lines. These changes did not correlate with changes in the expression of enzymes involved in the de novo biosynthesis or consumption of GSH. However, the cellular accumulation ratio for CQ indicated a decrease of free heme in the resistant parasites. Mutant forms of PfCRT expressed in oocytes of Xenopus laevis were able to transport GSH, while the sensitive wild-type form did not transport the tripeptide. The findings of this study suggest that in parasites bearing mutant PfCRT, GSH is transported into the digestive vacuole where it is able to contribute to resistance by degrading heme, before the tripeptide itself is degraded by peptidases inside the vacuole, consistent with the overall reduction of GSH levels in CQ resistant parasites

Development of a transgenic Plasmodium berghei line (Pb pfpkg) expressing the P. falciparum cGMP-dependent protein kinase, a novel antimalarial drug target.

With the inevitable selection of resistance to antimalarial drugs in treated populations, there is a need for new medicines to enter the clinic and new targets to progress through the drug discovery pipeline. In this study we set out to develop a transgenic rodent model for testing inhibitors of the Plasmodium falciparum cyclic GMP-dependent kinase in vivo. A model was needed that would allow us to investigate whether differences in amino acid sequence of this enzyme between species influences in vivo efficacy. Here we report the successful development of a transgenic P. berghei line in which the cyclic GMP-dependent protein kinase (PKG) was replaced by the P. falciparum orthologue. We demonstrate that the P. falciparum orthologue was able to functionally complement the endogenous P. berghei pkg gene throughout blood stage development and early sexual development. However, subsequent development in the mosquito was severely compromised. We show that this is due to a defect in the female lineage of the transgenic by using genetic crosses with both male and female deficient P. berghei lines. This defect could be due to expression of a female-specific target in the mosquito stages of P. berghei that cannot be phosphorylated by the P. falciparum kinase. Using a previously reported anti-coccidial inhibitor of the cyclic GMP-dependent protein kinase, we show no difference in in vivo efficacy between the transgenic and control P. berghei lines. This in vivo model will be useful for screening future generations of cyclic GMP-dependent protein kinase inhibitors and allowing us to overcome any species-specific differences in the enzyme primary sequence that would influence in vivo efficacy in the rodent model. The approach will also be applicable to in vivo testing of other antimalarial compounds where the target is known

ThePlasmodiumClass XIV Myosin, MyoB, Has a Distinct Subcellular Location in Invasive and Motile Stages of the Malaria Parasite and an Unusual Light Chain

Myosin B (MyoB) is one of the two short class XIV myosins encoded in the Plasmodium genome. Class XIV myosins are characterized by a catalytic “head,” a modified “neck,” and the absence of a “tail” region. Myosin A (MyoA), the other class XIV myosin in Plasmodium, has been established as a component of the glideosome complex important in motility and cell invasion, but MyoB is not well characterized. We analyzed the properties of MyoB using three parasite species as follows: Plasmodium falciparum, Plasmodium berghei, and Plasmodium knowlesi. MyoB is expressed in all invasive stages (merozoites, ookinetes, and sporozoites) of the life cycle, and the protein is found in a discrete apical location in these polarized cells. In P. falciparum, MyoB is synthesized very late in schizogony/merogony, and its location in merozoites is distinct from, and anterior to, that of a range of known proteins present in the rhoptries, rhoptry neck or micronemes. Unlike MyoA, MyoB is not associated with glideosome complex proteins, including the MyoA light chain, myosin A tail domain-interacting protein (MTIP). A unique MyoB light chain (MLC-B) was identified that contains a calmodulin-like domain at the C terminus and an extended N-terminal region. MLC-B localizes to the same extreme apical pole in the cell as MyoB, and the two proteins form a complex. We propose that MLC-B is a MyoB-specific light chain, and for the short class XIV myosins that lack a tail region, the atypical myosin light chains may fulfill that role

Synthesis and anti-protozoal activity of C2-substituted polyazamacrocycles

A focused library of C2-substituted-1,4,7,10-tetraazacyclododecanes was synthesised and the compounds were tested for their ability to kill trypanosome and malaria parasites. Several compounds showed significant in vitro activity and were selectively active against the parasites over human embryonic kidney cells used as a counter scree

Synthesis of novel benzamidine- and guanidine-derived polyazamacrocycles: Selective anti-protozoal activity for human African trypanosomiasis

Efficient synthetic routes have been developed for the preparation of two new polyazamacrocycles tagged with structural motifs recognised by the Trypanosoma brucei P2 aminopurine transporter. Biological testing of these compounds showed highly selective anti-protozoal activity against trypanosome

Apicoplast lipoic acid protein ligase B is not essential for Plasmodium falciparum

Lipoic acid (LA) is an essential cofactor of alpha-keto acid dehydrogenase complexes (KADHs) and the glycine cleavage system. In Plasmodium, LA is attached to the KADHs by organelle-specific lipoylation pathways. Biosynthesis of LA exclusively occurs in the apicoplast, comprising octanoyl-[acyl carrier protein]: protein N-octanoyltransferase (LipB) and LA synthase. Salvage of LA is mitochondrial and scavenged LA is ligated to the KADHs by LA protein ligase 1 (LplA1). Both pathways are entirely independent, suggesting that both are likely to be essential for parasite survival. However, disruption of the LipB gene did not negatively affect parasite growth despite a drastic loss of LA (&gt; 90%). Surprisingly, the sole, apicoplast-located pyruvate dehydrogenase still showed lipoylation, suggesting that an alternative lipoylation pathway exists in this organelle. We provide evidence that this residual lipoylation is attributable to the dual targeted, functional lipoate protein ligase 2 (LplA2). Localisation studies show that LplA2 is present in both mitochondrion and apicoplast suggesting redundancy between the lipoic acid protein ligases in the erythrocytic stages of P. falciparum.</p