Erythrocytic Adenosine Monophosphate as an Alternative Purine Source in Plasmodium falciparum

Abstract

Plasmodium falciparum is a purine auxotroph, salvaging purines from erythrocytes for synthesis of RNA and DNA. Hypoxanthine is the key precursor for purine metabolism in Plasmodium. Inhibition of hypoxanthine-forming reactions in both erythrocytes and parasites is lethal to cultured P. falciparum. We observed that high concentrations of adenosine can rescue cultured parasites from purine nucleoside phosphorylase and adenosine deaminase blockade but not when erythrocyte adenosine kinase is also inhibited. P. falciparum lacks adenosine kinase but can salvage AMP synthesized in the erythrocyte cytoplasm to provide purines when both human and Plasmodium purine nucleoside phosphorylases and adenosine deaminases are inhibited. Transport studies in Xenopus laevis oocytes expressing the P. falciparum nucleoside transporter PfNT1 established that this transporter does not transport AMP. These metabolic patterns establish the existence of a novel nucleoside monophosphate transport pathway in P. falciparum.Malaria is one of the leading causes of morbidity and mortality in the tropics with 300 -500 million clinical cases and 1.5-2.7 million deaths per year (1). Malaria is caused by protozoan parasites of the Plasmodium genus of which Plasmodium falciparum is responsible for most of the fatal cases. The parasite is transmitted between human hosts by Anopheles mosquitoes. Malaria has been treated for many years through chemotherapeutic and vector control strategies, but these have not prevented widespread occurrence. An alarming increase in both the resistance of malaria parasites to drug treatment and in mosquito vectors to insecticides has renewed the demand for the development of new chemotherapeutic strategies (2, 3). Advances in the knowledge of the metabolic and nutritional needs of the parasite offer new potential routes for chemotherapy. Because of the rapid rate of DNA synthesis during the 48-h intraerythrocytic growth phase, targeting purine metabolic pathways is a promising route for novel drug development.P. falciparum is a purine auxotroph, salvaging host cell purines for synthesis of cofactors and nucleic acids (4, 5). Purine nucleosides and nucleobases can be transported across the parasite plasma membrane by the PfNT1 2 transporter (Fig. 1). The mechanisms by which Plasmodium salvages purines during the intraerythrocytic cycle are diverse both with regard to primary sources and to routes of interconversion (Fig. 1). Hypoxanthine is the key precursor of other purines in Plasmodium metabolism and is commonly used as a nutritional supplement in malarial culture media. A key source of hypoxanthine in vivo is from the erythrocyte purine pool where ATP is in dynamic metabolic exchange with hypoxanthine via ADP, AMP, IMP, inosine, and adenosine (5). In human erythrocytes, adenosine is efficiently salvaged by adenosine kinase (hAK). This allows human erythrocytes to utilize serum adenosine to maintain erythrocyte ATP levels. In P. falciparum, adenosine is salvaged by conversion to hypoxanthine using adenosine deaminas...

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

“…Soluble metabolites were extracted from the lysate by treatment with 0.5 M HClO 4 for 30 min on ice using published protocols (16). Samples were then neutralized with 5 M KOH and centrifuged to remove the precipitate.…”

Transmembrane Segment 11 Appears to Line the Purine Permeation Pathway of the Plasmodium falciparum Equilibrative Nucleoside Transporter 1 (PfENT1)

“…PfENT1 Plasmid and Site-specific Mutations-For these experiments, we used the PfENT1-pXOON plasmid containing the P. falciparum 3D7 strain pfent1 gene in the pXOON plasmid (52) that we used previously (53,16). The PfENT1-pXOON plasmid was linearized with NheI, and capped mRNA was prepared as described previously (16).…”

Characterization of an ecto-5′-nucleotidase activity present on the cell surface of Tritrichomonas foetus

“…Recently, it was demonstrated that Plasmodium falciparum is able to transport nucleotides, such as, 5 AMP from extracellular medium to inside the cell (Cassera et al, 2008). The presence of a 5 nucleotidase activity in the plasma membrane fraction 9.3-fold and 10.6-fold higher than cellular homogenate and soluble fraction, respectively ( Fig.…”