The Methylerythritol Phosphate Pathway Is Functionally Active in All Intraerythrocytic Stages of Plasmodium falciparum

Abstract

Malaria is one of the leading causes of morbidity and mortality in the tropics, with 300 to 500 million clinical cases and 1.5 to 2.7 million deaths per year. Nearly all fatal cases are caused by Plasmodium falciparum. The resistance of this parasite to conventional antimalarial drugs such as chloroquine is growing at an alarming rate and therefore new efficient drugs are urgently needed (1-3).In all organisms studied so far, the biosynthesis of isoprenoids such as dolichol, cholesterol, and ubiquinones depends on the condensation of the different numbers of isopentenyl diphosphate (IPP) 1 and dimethylallyl diphosphate units. In mammals and fungi, these units are derived from the classical mevalonate pathway (4). However, in higher plants, in several algae, in some eubacteria, and in P. falciparum the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway was described as the alternative non-mevalonate pathway for the synthesis of IPP (for reviews, see Refs. 5-10). This pathway starts with the condensation of pyruvate and glyceraldehyde 3-phosphate, which yields 1-deoxy-D-xylulose-5-phosphate (DOXP) as a key metabolite (11-17). The DOXP reductoisomerase then catalyzes the simultaneous intramolecular rearrangement and reduction of DOXP to form MEP (18 -22). The activity of this enzyme is specifically inhibited by fosmidomycin (23). Several reaction steps are necessary for the conversion of MEP to IPP. The downstream intermediates of MEP for this pathway are: 4-(cytidine-5-diphospho)-2-C-methyl-D-erythritol (CDP-ME) (24), 4-(cytidine-5-diphospho)-2-C-methyl-D-erythritol-2-phosphate (CDP-MEP), 2-C-methyl-D-erythritol-2,4-cyclodiphosphate (ME-2,4-cPP) (25, 26), and 4-hydroxy-3-methylbut-2-enyl pyrophosphate (27-31). IPP and dimethylallyl diphosphate are synthesized through independent routes in the late steps of the non-mevalonate pathway (32). These units are used for the biosynthesis of ubiquinones and dolichols, and for the prenylation of proteins and other products (33)(34)(35).Based on the sequence data provided by the malaria genome project (plasmodb.org), Jomaa and co-workers (21) identified two genes in P. falciparum that encode key enzymes of the MEP pathway: 1-deoxy-D-xylulose-5-phosphate synthase and 1-deoxy-D-xylulose-5-phosphate reductoisomerase. They also demonstrated that an amino-terminal signal sequence in 1-de- 1 The abbreviations and trivial names used are: IPP, isopentenyl diphosphate; MEP, 2-C-methyl-D-erythritol-4-phosphate; DOXP, 1-deoxy-D-xylulose-5-phosphate; DOX, 1-deoxy-D-xylulose; CDP-ME, 4-(cytidine-5-diphospho)-2-C-methyl-D-erythritol; CDP-MEP, 4-(cytidine-5-diphospho)-2-C-methyl-D-erythritol-2-phosphate; ME-2,4-cPP, 2-Cmethyl-D-erythritol-2,4-cyclodiphosphate; HPLC, high-performance liquid chromatography; ESI-QTOF-MS, ESI-quadrupole time-of-flight mass spectrometry; Q n , Coenzyme Q n .

Effect of fosmidomycin on metabolic and transcript profiles of the methylerythritol phosphate pathway in Plasmodium falciparum

“…Two independent experiments using [1-14 C]sodium acetate as a metabolic precursor were conducted to monitor each intermediate of the MEP pathway, dolichols and ubiquinones for each developmental stage. [1-14 C]sodium acetate is well incorporated into the MEP pathway intermediates in P. falciparum (Cassera et al 2004) instead of [1-14 C]pyruvic acid or [2-14 C]pyruvic acid, which is not incorporated by blood-stage P. falciparum (Cranmer et al 1995, Elliott et al 2001. Two cycles after sorbitol synchronization, cultures in ring, trophozoite or schizont stages with approximately 10% parasitemia, untreated or treated with 1 µM fosmidomycin for 31 h, were labeled with 6.25 µCi/ml of [1-14 C]sodium acetate (56 mCi/mmol, Amersham Biosciences) in the last 17 h, and recovered for analysis.…”

“…Two cycles after sorbitol synchronization, cultures in ring, trophozoite or schizont stages with approximately 10% parasitemia, untreated or treated with 1 µM fosmidomycin for 31 h, were labeled with 6.25 µCi/ml of [1-14 C]sodium acetate (56 mCi/mmol, Amersham Biosciences) in the last 17 h, and recovered for analysis. Seventeen hours of exposure time is the minimum time to detect 14 C incorporation into the MEP pathway intermediates (Cassera et al 2004). The parasites were isolated by treatment with 0.1% (w/v) saponin for 5 min, followed by two washes with phosphate-buffered saline (PBS), pH 7.2, and stored in liquid N 2 for subsequent HPLC analysis.…”

“…Recent field trials in humans have also demonstrated the effectiveness of fosmidomycin in the treatment of human malarial infections, but it has to be administered for more than four days when used alone (Missinou et al 2002, Borrmann et al 2005. Recently, biochemical and mass spectrometric analyses revealed that the MEP pathway is functionally active in all intraerythrocytic stages of P. falciparum (Cassera et al 2004).In this study we characterized the effect of fosmidomycin on the metabolic levels of each intermediate of the MEP pathway as well as dolichol and ubiquino- nes in ring, trophozoite and schizont stages of P. falciparum parasites and correlated these to the steadystate MEP enzyme transcript levels under drug pressure. …”

“…Recent field trials in humans have also demonstrated the effectiveness of fosmidomycin in the treatment of human malarial infections, but it has to be administered for more than four days when used alone (Missinou et al 2002, Borrmann et al 2005. Recently, biochemical and mass spectrometric analyses revealed that the MEP pathway is functionally active in all intraerythrocytic stages of P. falciparum (Cassera et al 2004).…”

“…Aliquots of each extract were monitored for radioactivity with a Beckman LS 5000 TD β-counter. Metabolic analyses by HPLC of DOXP, pyridoxal 5'-phosphate (PNP), MEP, CDP-ME, CDP-MEP, MEcPP, dolichols and ubiquinones were carried out as described previously (Cassera et al 2004). …”