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

Abstract

Key words: Plasmodium falciparum -malaria -fosmidomycin -isoprenoid biosynthesis -real time polymerase chain reaction Malaria is a leading cause of morbidity and mortality in the tropical regions, with 300 to 500 million clinical cases and 1.5 to 2.7 million deaths per year (Snow et al. 2005). With the availability of the complete genome sequence from Plasmodium falciparum, increasing attention has focused on transcript profiling and proteomic analyses of the parasite stages responsible for severe disease and pathogenesis in humans (Florens et al. 2002, Bozdech et al. 2003, Le Roch et al. 2003, Nirmalan et al. 2004, Llinas et al. 2006.Two different biosynthetic routes are used to produce isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) for isoprenoid biosynthesis including ubiquinones, dolichols and the prenylation of proteins (Sacchettini & Poulter 1997, Sinensky 2000, Barkovich & Liao 2001. In mammals, plants (cytoplasm), fungi, some bacteria and several protozoa, the isoprenic units are derived from the classical mevalonate pathway (Goldstein & Brown 1990). In plastids of plants, several algae, eubacteria, cyanobacteria and apicomplexa (apicoplast), the 2C-methyl-D-erythritol-4-phosphate (MEP) pathway produces IPP and DMAPP (Rohmer 1999). The MEP pathway starts with the condensation of pyruvate and glyceraldehyde-3-phosphate (GAP), which yields 1-deoxy-D-xylulose-5-phosphate (DOXP) catalyzed by DOXP synthase (DXS); for reviews see references (Lichtenthaler 1999, Rohmer 1999, Eisenreich et al. 2004, Rodriguez-Concepcion 2004. DOXP reductoisomerase (DXR) then catalyzes the intramolecular rearrangement and reduction of DOXP to MEP. The activity of this enzyme is specifically inhibited by fosmidomycin (Kuzuyama et al. 1998). Several subsequent reaction steps are necessary for the conversion of MEP to IPP and DMAPP (Fig. 1).Discovery of the MEP pathway for isoprenoid biosynthesis in P. falciparum revealed several antimalarial drug targets (Jomaa et al. 1999). Jomaa and co-workers demonstrated that fosmidomycin and its derivate FR900098, are able to inhibit the growth of P. falciparum in culture and to cure mice infected with the related malaria parasite, P. vinckei (Jomaa et al. 1999). 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. MATERIALS AND METHODSExperimental design -Three characteristic developmental sta...

Isoprenoid Biosynthesis in Plasmodium falciparum

“…Labeling experiments demonstrate that these dolichols are formed from FPP and GPP, respectively (123,124). As expected, dolichol synthesis is also sensitive to fosmidomycin treatment (37 (125). The parasite is sensitive to known inhibitors of GPI synthesis, such as the mannose analogue 2-deoxyglucose (126,127).…”

“…Analysis of MEP pathway intermediates in bacteria and P. falciparum has established that fosmidomycin reduces the intracellular levels of downstream MEP pathway metabolites and isoprenoid products (35)(36)(37). In addition, the growth inhibitory effects of fosmidomycin are chemically rescued in bacteria and malaria parasites through supplementation of the medium with IPP or unphosphorylated isoprenols (farnesol and geranylgeraniol).…”

“…Studies in Arabidopsis thaliana have identified an RNA processing protein, Rif10, which posttranscriptionally regulates the levels of MEP pathway enzymes (143). However, studies in P. falciparum have indicated that the transcript levels of MEP pathway enzymes do not change significantly upon chemical inhibition of the MEP pathway, suggesting that modulating transcript levels is likely not a primary mechanism of MEP pathway regulation in the parasite (37). In the asexual intraerythrocytic cycle, the transcript levels of MEP pathway genes peak in the late trophozoite stage (144).…”

Antiapicoplast and Gametocytocidal Screening To Identify the Mechanisms of Action of Compounds within the Malaria Box

“…A diminished membrane potential is consistent with the role of the apicoplast isoprenoid pathway in ubiquinone biosynthesis. It has been demonstrated that FOS inhibits ubiquinone biosynthesis (17,32). Ubiquinones participate in the electron transport chain, and their biosynthesis relies on the MEP pathway to provide precursors for the isoprene side chain that anchors them into the inner membrane of the mitochondria.…”

Isoprenoid Precursor Biosynthesis Is the Essential Metabolic Role of the Apicoplast during Gametocytogenesis in Plasmodium falciparum

“…Our results showed that MEP intermediates are present in gametocytes (Table 1) and that isoprenoids are essential for gametocytogenesis, suggesting that the loss of FOS and MMV008138 activity in gametocytes, especially during the early stages (38,48), may be due to lack of transport mechanisms for these inhibitors or unknown metabolic regulation factors. The apicoplast is the sole source of isoprenoid precursors through the MEP pathway during the intraerythrocytic stages, as the human erythrocytes do not have an active mevalonate pathway to synthesize IPP (27,(49)(50)(51). In addition, it is unlikely that IPP can be scavenged from human plasma at the required concentration (200 M) because it has not been detected in the human serum metabolome (52).…”