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Leishmaniasis is a neglected disease caused by a trypanosomatid protozoan of the genus Leishmania. Most drugs used to treat leishmaniasis are highly toxic, and the emergence of drug-resistant strains has been observed. Therefore, new therapeutic targets against leishmaniasis are required. Several isoprenoid compounds, including dolichols or ubiquinones, have been shown to be important for cell viability and proliferation in various trypanosomatid species. Here, we detected the biosynthesis of tocopherol in Leishmania (L.) amazonensis promastigotes in vitro through metabolic labelling with [1-(n)-3H]-phytol. Subsequently, we confirmed the presence of vitamin E in the parasite by gas chromatography-mass spectrometry. Treatment with usnic acid or nitisinone, inhibitors of precursors of vitamin E synthesis, inhibited growth of the parasite in a concentration-dependent manner. This study provides the first evidence of tocopherol biosynthesis in a trypanosomatid and suggests that inhibitors of the enzyme 4-hydroxyphenylpyruvate dioxygenase may be suitable for use as antileishmanial compounds. Database The amino acid sequence of a conserved hypothetical protein [Leishmania mexicana MHOM/GT/2001/U1103] has been deposited in GenBank (CBZ28005.1).

Atovaquone (AV) acts on the malaria parasite by competing with ubiquinol (UQH(2)) for its union to the mitochondrial bc(1) complex, preventing the ubiquinone-8 and ubiquinone-9 (UQ-8 and UQ-9) redox recycling, which is a necessary step in pyrimidine biosynthesis. This study focused on UQ biosynthesis in Plasmodium falciparum and adopted proof-of-concept research to better elucidate the mechanism of action of AV and improve its efficacy. Initially, UQ biosynthesis was evaluated using several radioactive precursors and chromatographic techniques. This methodology was suitable for studying the biosynthesis of both UQ homologs and its redox state. Additionally, the composition of UQ was investigated in parasites cultivated at different oxygen saturations or in the presence of AV. AV affected the redox states of both UQ-8 and UQ-9 homologs by increasing the levels of the respective reduced forms. Conversely, low-oxygen environments specifically inhibited UQ-9 biosynthesis and increased the antimalarial efficacy of AV. These findings encouraged us to investigate the biological importance and the potential of UQ biosynthesis as a drug target based on its inhibition by 4-nitrobenzoate (4-NB), a 4-hydroxybenzoate (4-HB) analog. 4-NB effectively inhibits UQ biosynthesis and enhances the effects of AV on parasitic growth and respiration rate. Although 4-NB itself exhibits poor antimalarial activity, its 50% inhibitory concentration (IC(50)) value increased significantly in the presence of a soluble UQ analog, p-aminobenzoic acid (pABA), or 4-HB. These results indicate the potential of AV combined with 4-NB as a novel therapy for malaria and other diseases caused by AV-sensitive pathogens.

Malaria, an infectious disease that kills more than 438,000 people per year worldwide, is a major public health problem. The emergence of strains resistant to conventional therapeutic agents necessitates the discovery of new drugs. We previously demonstrated that various substances, including terpenes, have antimalarial activity in vitro and in vivo. Nerolidol is a sesquiterpene present as an essential oil in several plants that is used in scented products and has been approved by the US Food and Drug Administration as a food-flavouring agent. In this study, the antimalarial activity of nerolidol was investigated in a mouse model of malaria. Mice were infected with Plasmodium berghei ANKA and were treated with 1000 mg/kg/dose nerolidol in two doses delivered by the oral or inhalation route. In mice treated with nerolidol, parasitaemia was inhibited by99% (oral) and80% (inhalation) until 14 days after infection (P 0.0001). On Day 30 post-infection, the survival rate of orally treated mice was 90% compared with 16% in controls (P 0.0001). In contrast, inhalation-treated mice showed a survival rate of 50% vs. 42% in controls (P 0.05). The toxicity of nerolidol administered by either route was not significant, whilst genotoxicity was observed only at the highest dose tested. These results indicate that combined use of nerolidol and other drugs targeting different points of the same isoprenoid pathway may be an effective treatment for malaria.

Human parasitic protozoa cause a large number of diseases worldwide and, for some of these diseases, there are no effective treatments to date, and drug resistance has been observed. For these reasons, the discovery of new etiological treatments is necessary. In this sense, parasitic metabolic pathways that are absent in vertebrate hosts would be interesting research candidates for the identification of new drug targets. Most likely due to the protozoa variability, uncertain phylogenetic origin, endosymbiotic events, and evolutionary pressure for adaptation to adverse environments, a surprising variety of prenylquinones can be found within these organisms. These compounds are involved in essential metabolic reactions in organisms, for example, prevention of lipoperoxidation, participation in the mitochondrial respiratory chain or as enzymatic cofactors. This review will describe several prenylquinones that have been previously characterized in human pathogenic protozoa. Among all existing prenylquinones, this review is focused on ubiquinone, menaquinone, tocopherols, chlorobiumquinone, and thermoplasmaquinone. This review will also discuss the biosynthesis of prenylquinones, starting from the isoprenic side chains to the aromatic head group precursors. The isoprenic side chain biosynthesis maybe come from mevalonate or non-mevalonate pathways as well as leucine dependent pathways for isoprenoid biosynthesis. Finally, the isoprenic chains elongation and prenylquinone aromatic precursors origins from amino acid degradation or the shikimate pathway is reviewed. The phylogenetic distribution and what is known about the biological functions of these compounds among species will be described, as will the therapeutic strategies associated with prenylquinone metabolism in protozoan parasites.

Farnesyl diphosphate synthase/geranylgeranyl diphosphate synthase (FPPS/GGPPS) is a key enzyme in the synthesis of isoprenic chains. Risedronate, a bisphosphonate containing nitrogen (N-BP), is a potent inhibitor of blood stage Plasmodium. Here, we show that P. falciparum parasites overexpressing FPPS/GGPPS are more resistant to risedronate, suggesting that this enzyme is an important target, and bisphosphonate analogues can be used as potential antimalarial drugs.

Background Plasmodium falciparum is sensitive to oxidative stress in vitro and in vivo, and many drugs such as artemisinin, chloroquine and cercosporin interfere in the parasite’s redox system. To minimize the damage caused by reactive radicals, antioxidant enzymes and their substrates found in parasites and in erythrocytes must be functionally active. It was shown that P. falciparum synthesizes vitamin E and that usnic acid acts as an inhibitor of its biosynthesis. Vitamin E is a potent antioxidant that protects polyunsaturated fatty acids from lipid peroxidation, and this activity can be measured by detecting its oxidized product and by evaluating reactive oxygen species (ROS) levels. Results Here, we demonstrated that ROS levels increased in P. falciparum when vitamin E biosynthesis was inhibited by usnic acid treatment and decreased to basal levels if exogenous vitamin E was added. Furthermore, we used metabolic labelling to demonstrate that vitamin E biosynthesized by the parasite acts as an antioxidant since we could detect its radiolabeled oxidized product. The treatment with chloroquine or cercosporin of the parasites increased the ratio between α-tocopherolquinone and α-tocopherol. Conclusions Our findings demonstrate that vitamin E produced endogenously by P. falciparum is active as an antioxidant, probably protecting the parasite from the radicals generated by drugs. Electronic supplementary material The online version of this article (10.1186/s13071-017-2402-3) contains supplementary material, which is available to authorized users.

The development of new drugs is one of the strategies to control malaria. Isoprenoid biosynthesis in Plasmodium falciparum is an essential pathway for parasite survival, and is therefore a potential target for new antimalarial drugs. Indeed, plant-derived secondary metabolites, such as terpenes, exhibit antimalarial activity in vitro by inhibiting isoprenoid biosynthesis in P. falciparum. In this study, the in vitro antiplasmodial activity of perillyl alcohol (POH) was evaluated, along with its in vitro toxicity and its effect on the isoprenylation process. In addition, the efficacy of intranasally administered POH in preventing Plasmodium berghei ANKA-induced experimental cerebral malaria (ECM) was determined. The 50% inhibitory concentrations of POH for 3D7 and K1 P. falciparum were 4.8 µM and 10.4 µM, respectively. POH inhibited farnesylation of 20-37 kDa proteins in P. falciparum (3D7), but no toxic effects in Vero cells were observed. A 500 mg/kg/d dose of POH had no effect on P. berghei ANKA parasitaemia, but showed marked efficacy in preventing ECM development (70% survival compared with 30% for untreated animals). This effect was associated with the downregulation of cerebrovascular inflammation and damage, with marked decreases in brain leucocyte accumulation and the incidence of brain microhaemorrhage. POH also downregulated interleukin (IL)-10, IL-6, tumour necrosis factor-α, interferon-γ, IL-12 and monocyte chemoattractant protein-1 levels in the brain and spleen. In conclusion, POH shows antiplasmodial activity in vitro and, despite there being no evidence of antiplasmodial activity in vivo following intranasal administration, POH prevented cerebrovascular inflammation/damage and expression of pro-inflammatory cytokines.

In Plasmodium falciparum, the formation of isopentenyl diphosphate and dimethylallyl diphosphate, central intermediates in the biosynthesis of isoprenoids, occurs via the methylerythritol phosphate (MEP) pathway. Fosmidomycin is a specific inhibitor of the second enzyme of the MEP pathway, 1-deoxy-D-xylulose-5-phosphate reductoisomerase. We analyzed the effect of fosmidomycin on the levels of each intermediate and its metabolic requirement for the isoprenoid biosynthesis, such as dolichols and ubiquinones, throughout the intraerythrocytic cycle of P. falciparum. The steady-state RNA levels of the MEP pathway-associated genes were quantified by real-time polymerase chain reaction and correlated with the related metabolite levels. Our results indicate that MEP pathway metabolite peak precede maximum transcript abundance during the intraerythrocytic cycle. Fosmidomycin-treatment resulted in a decrease of the intermediate levels in the MEP pathway as well as in ubiquinone and dolichol biosynthesis. The MEP pathway associated transcripts were modestly altered by the drug, indicating that the parasite is not strongly responsive at the transcriptional level. This is the first study that compares the effect of fosmidomycin on the metabolic and transcript profiles in P. falciparum, which has only the MEP pathway for isoprenoid biosynthesis.

Malaria is a tropical disease with significant morbidity and mortality. A better understanding of the metabolism of its most important etiological agent, Plasmodium falciparum, is paramount to the development of better treatment and other mitigation measures. Farnesyldiphosphate synthase/geranylgeranyldiphosphate synthase (FPPS/GGPPS) is a key enzyme in the synthesis of isoprenic chains present in many essential structures. In P. falciparum, as well as a handful of other organisms, FPPS/GGPPS has been shown to be a bifunctional enzyme. By genetic tagging and microscopy, we observed a changing localization of FPPS/GGPPS in blood stage parasites. Given the great importance of alternative splicing and other transcriptional phenomena in gene regulation and the generation of protein diversity, we have investigated the processing of the FPPS/GGPPS transcript in P. falciparum by high-throughput sequencing methods in four time-points along the intraerythrocytic cycle of P. falciparum. We have identified levels of transcript diversity an order of magnitude higher than previously observed in this organism, as well as a few stage-specific splicing events. Our data suggest that alternative splicing in P. falciparum is an important feature for gene regulation and the generation of protein diversity.

Nerolidol is a naturally occurring sesquiterpene found in the essential oils of many types of flowers and plants. It is frequently used in cosmetics, as a food flavoring agent, and in cleaning products. In addition, nerolidol is used as a skin penetration enhancer for transdermal delivery of therapeutic drugs. However, nerolidol is hemolytic at low concentrations. A simple and fast GC–MS method was developed for preliminary quantification and assessment of biological interferences of nerolidol in mouse plasma after oral dosing. Calibration curves were linear in the concentration range of 0.010–5 μg/mL nerolidol in mouse plasma with correlation coefficients (r) greater than 0.99. Limits of detection and quantification were 0.0017 and 0.0035 μg/mL, respectively. The optimized method was successfully applied to the quantification of nerolidol in mouse plasma.

The increasing resistance of malaria parasites to almost all available drugs calls for the characterization of novel targets and the identification of new compounds. Carotenoids are polyisoprenoids from plants, algae, and some bacteria, and they are biosynthesized by Plasmodium falciparum but not by mammalian cells. Biochemical and reverse genetics approaches were applied to demonstrate that phytoene synthase (PSY) is a key enzyme for carotenoid biosynthesis in P. falciparum and is essential for intraerythrocytic growth. The known PSY inhibitor squalestatin reduces biosynthesis of phytoene and kills parasites during the intraerythrocytic cycle. PSY-overexpressing parasites showed increased biosynthesis of phytoene and its derived product phytofluene and presented a squalestatin-resistant phenotype, suggesting that this enzyme is the primary target of action of this drug in the parasite.

We performed reverse-phase thin-layer chromatography and reverse-phase high-performance liquid chromatography (RP-HPLC) analysis of polyisoprenoids released by sulfonium-salt cleavage with methyl iodide from Plasmodium falciparum proteins labeled with [3H]FPP or [3H]GGPP and showed that a dolichol of 11 isoprene units is bound to 21–28-kDa protein clusters from trophozoite and schizont stages. The dolichol structure was confirmed by electrospray-ionization mass spectrometry analysis. Treatment with protein synthesis inhibitors and RP-HPLC analysis of the proteolytic digestion products from parasite proteins labeled with [35S]cysteine and [3H]FPP showed that the attachment of dolichol to protein is a post-translational event and probably occurs via a covalent bond to cysteine residues.

Isoprenoids play important roles in all living organisms as components of structural cholesterol, steroid hormones in mammals, carotenoids in plants, and ubiquinones. Significant differences occur in the length of the isoprenic side chains of ubiquinone between different organisms, suggesting that different enzymes are involved in the synthesis of these side chains. Whereas in Plasmodium falciparum the isoprenic side chains of ubiquinone contain 7–9 isoprenic units, 10-unit side chains are found in humans. In a search for the P. falciparum enzyme responsible for the biosynthesis of isoprenic side chains attached to the benzoquinone ring of ubiquinones, we cloned and expressed a putative polyprenyl synthase. Polyclonal antibodies raised against the corresponding recombinant protein confirmed the presence of the native protein in trophozoite and schizont stages of P. falciparum. The recombinant protein, as well as P. falciparum extracts, showed an octaprenyl pyrophosphate synthase activity, with the formation of a polyisoprenoid with eight isoprenic units, as detected by reverse-phase HPLC and reverse-phase TLC, and confirmed by electrospray ionization and tandem MS analysis. The recombinant and native versions of the enzyme had similar Michaelis constants with the substrates isopentenyl pyrophosphate and farnesyl pyrophosphate. The recombinant enzyme could be competitively inhibited in the presence of the terpene nerolidol. This is the first report that directly demonstrates an octaprenyl pyrophosphate synthase activity in parasitic protozoa. Given the rather low similarity of the P. falciparum enzyme to its human counterpart, decaprenyl pyrophosphate synthase, we suggest that the identified enzyme and its recombinant version could be exploited in the screening of novel drugs.

Two genes encoding the enzymes 1-deoxy-D-xylulose-5-phosphate synthase and 1-deoxy-D-xylulose-5-phosphate reductoisomerase have been recently identified, suggesting that isoprenoid biosynthesis in Plasmodium falciparum depends on the methylerythritol phosphate (MEP) pathway, and that fosmidomycin could inhibit the activity of 1-deoxy-D-xylulose-5-phosphate reductoisomerase. The metabolite 1-deoxy-D-xylulose-5-phosphate is not only an intermediate of the MEP pathway for the biosynthesis of isopentenyl diphosphate but is also involved in the biosynthesis of thiamin (vitamin B1) and pyridoxal (vitamin B6) in plants and many microorganisms. Herein we report the first isolation and characterization of most downstream intermediates of the MEP pathway in the three intraerythrocytic stages of P. falciparum. These include, 1-deoxy-D-xylulose-5-phosphate, 2-C-methyl-D-erythritol-4-phosphate, 4-(cytidine-5-diphospho)-2-C-methyl-D-erythritol, 4-(cytidine-5-diphospho)-2-C-methyl-D-erythritol-2-phosphate, and 2-C-methyl-D-erythritol-2,4-cyclodiphosphate. These intermediates were purified by HPLC and structurally characterized via biochemical and electrospray mass spectrometric analyses. We have also investigated the effect of fosmidomycin on the biosynthesis of each intermediate of this pathway and isoprenoid biosynthesis (dolichols and ubiquinones). For the first time, therefore, it is demonstrated that the MEP pathway is functionally active in all intraerythrocytic forms of P. falciparum, and de novo biosynthesis of pyridoxal in a protozoan is reported. Its absence in the human host makes both pathways very attractive as potential new targets for antimalarial drug development.

Little is known about isoprenoid biosynthesis in parasitic protozoa. The presence of dolichol and isoprenylated proteins has been detected in Plasmodium falciparum, but no studies are available about the biosynthesis of the isoprenic side chain attached to the benzoquinone ring of coenzyme Q. In the present study, using metabolic labelling with different intermediates, we demonstrated the presence of an active isoprenoid pathway for the biosynthesis of the isoprenic chain of coenzyme Q. Our results show that P. falciparum is able to synthesize different homologs (coenzyme Q(8) and coenzyme Q(9)), depending on the given intermediate. Parasites treated with nerolidol at doses 2.2 times below the IC(50) showed a decreased ability to synthesize the isoprenic chain attached to coenzyme Q at all intraerythrocytic stages. Treatment with nerolidol arrested development of the intraerythrocytic stages of the parasites, indicating that the drug may have an antimalarial potential.

N-glycosylation of proteins is required for the intra-erythrocytic schizogony of Plasmodium falciparum. In eukaryotic cells, this process involves the transfer of oligosaccharides from a dolichyl pyrophosphate derivative to asparagine residues. We have identified dolichol, dolichyl phosphate and dolichyl pyrophosphate species of 11 and 12 isoprenoid residues by metabolic labelling with [3H]farnesyl pyrophosphate, [3H]geranylgeranyl pyrophosphate and [14C]acetate in the different intra-erythrocytic stages of P. falciparum. This is the first demonstration of short-chain dolichols in the phylum Apicomplexa. The results demonstrate the presence of an active isoprenoid pathway in the intra-erythrocytic stages of P. falciparum. Parasites treated with mevastatin, a 3-hydroxy-3-methylglutaryl-CoA reductase inhibitor, show depressed biosynthesis of dolichol, dolichyl phosphate and isoprenoid pyrophosphate. This effect is observed in all intra-erythrocytic stages of the parasite life cycle, but is most pronounced in the ring stage. N-linked glycosylation of proteins was inhibited in the ring and young-trophozoite stages after mevastatin treatment of parasite cultures. Therefore the isoprenoid pathway may represent a different approach to the development of new anti-malarial drugs.

Background Isoprenoids are the most diverse and abundant group of natural products. In Plasmodium falciparum, isoprenoid synthesis proceeds through the methyl erythritol diphosphate pathway and the products are further metabolized by farnesyl diphosphate synthase (FPPS), turning this enzyme into a key branch point of the isoprenoid synthesis. Changes in FPPS activity could alter the flux of isoprenoid compounds downstream of FPPS and, hence, play a central role in the regulation of a number of essential functions in Plasmodium parasites. Methods The isolation and cloning of gene PF3D7_18400 was done by amplification from cDNA from mixed stage parasites of P. falciparum. After sequencing, the fragment was subcloned in pGEX2T for recombinant protein expression. To verify if the PF3D7_1128400 gene encodes a functional rPfFPPS protein, its catalytic activity was assessed using the substrate [4-14C] isopentenyl diphosphate and three different allylic substrates: dimethylallyl diphosphate, geranyl diphosphate or farnesyl diphosphate. The reaction products were identified by thin layer chromatography and reverse phase high-performance liquid chromatography. To confirm the product spectrum formed of rPfFPPS, isoprenic compounds were also identified by mass spectrometry. Apparent kinetic constants K M and V max for each substrate were determined by Michaelis–Menten; also, inhibition assays were performed using risedronate. Results The expressed protein of P. falciparum FPPS (rPfFPPS) catalyzes the synthesis of farnesyl diphosphate, as well as geranylgeranyl diphosphate, being therefore a bifunctional FPPS/geranylgeranyl diphosphate synthase (GGPPS) enzyme. The apparent K M values for the substrates dimethylallyl diphosphate, geranyl diphosphate and farnesyl diphosphate were, respectively, 68 ± 5 μM, 7.8 ± 1.3 μM and 2.06 ± 0.4 μM. The protein is expressed constitutively in all intra-erythrocytic stages of P. falciparum, demonstrated by using transgenic parasites with a haemagglutinin-tagged version of FPPS. Also, the present data demonstrate that the recombinant protein is inhibited by risedronate. Conclusions The rPfFPPS is a bifunctional FPPS/GGPPS enzyme and the structure of products FOH and GGOH were confirmed mass spectrometry. Plasmodial FPPS represents a potential target for the rational design of chemotherapeutic agents to treat malaria.

Although the existence of O-linked oligosaccharide residues in glycoproteins of Plasmodium falciparum has been shown, the existence of N-linked glycoproteins is still a matter of controversy and skepticism. This report demonstrates the unequivocal presence of N-linked glycoproteins in P. falciparum, principally in the ring and young trophozoite stages of the intraerythrocytic cycle. These glycoproteins lose their capacity to bind to concanavalin A-Sepharose after treatment of cultures with tunicamycin under conditions that do not affect protein synthesis. When the glycoproteins were treated with N-Glycanase®, oligosaccharides were released. It was possible to identify an N-linked glycoprotein of >200 kDa in the ring stage and also N-linked glycoproteins in the range of 200–30 kDa in the trophozoite stage. Treatment of trophozoites with 12 μM tunicamycin inhibited differentiation to the schizont stage. To our knowledge, this is the first report in the literature unequivocally showing N-linked glycoproteins in trophozoites of P. falciparum as well as their importance for the differentiation of the intraerythrocytic stages of this parasite.

Malaria continues to be one of the major threats to human health, affecting 300-500 million people and causing the death of approximately 1 million individuals per year, mostly children under 5 years of age (WHO 2010b). Human malaria is caused by five species of the genus Plasmodium, namely Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae and Plasmodium knowlesi, whereas the latter is found exclusively in the Southeast Asian region (Cox-Singh & Singh 2008). Current estimates suggest that approximately 2.4 billion people are at risk of stable or unstable Plasmodium falciparum transmission, similar global estimates are also available for P. vivax, and while there is considerably less mortality attributed to this species, its geographical reach is far greater. An estimated 2.9 billion people are at risk for vivax malaria, with an estimated 80 million to 300 million clinical cases annually (Guerra et al., 2010). These global estimates are a direct result of an increasing ability to collate and assimilate large data sets that also allow the monitoring of trends in malaria incidence and parasite prevalence. P. falciparum is strongly associated with a potentially fatal form of the disease, although recent reports indicate an underestimation of the severity of P. vivax infections (Alexandre et al., 2010). Efforts were made to eradicate malaria and although these were successful over large geographical areas, they did not succeed in tropical Africa or in many parts of Asia. In the past few years, malaria has once again attracted more attention partly because of increasing recognition that the malaria prevalence in sub-Saharan Africa has increased during the past decade. The main cause of the worsened malaria situation recorded in recent years has been the spread of drug-resistant parasites, which has led to rising malariaassociated mortality, especially in east Africa.

The increasing resistance of malarial parasites to almost all available drugs calls for the identification of new compounds and the detection of novel targets. Here, we establish the antimalarial activities of risedronate, one of the most potent bisphosphonates clinically used to treat bone resorption diseases, against blood stages of Plasmodium falciparum (50% inhibitory concentration [IC 50 ] of 20.3 ± 1.0 μM). We also suggest a mechanism of action for risedronate against the intraerythrocytic stage of P. falciparum and show that protein prenylation seems to be modulated directly by this drug. Risedronate inhibits the transfer of the farnesyl pyrophosphate group to parasite proteins, an effect not observed for the transfer of geranylgeranyl pyrophosphate. Our in vivo experiments further demonstrate that risedronate leads to an 88.9% inhibition of the rodent parasite Plasmodium berghei in mice on the seventh day of treatment; however, risedronate treatment did not result in a general increase of survival rates.

The development of new drugs is one strategy for malaria control. Biochemical pathways localised in the apicoplast of the parasite, such as the synthesis of isoprenic precursors, are excellent targets because they are different or absent in the human host. Isoprenoids are a large and highly diverse group of natural products with many functions and their synthesis is essential for the parasite's survival. During the last few years, the genes, enzymes, intermediates and mechanisms of this biosynthetic route have been elucidated. In this review, we comment on some aspects of the methylerythritol phosphate pathway and discuss the presence of diverse isoprenic products such as dolichol, ubiquinone, carotenoids, menaquinone and isoprenylated proteins, which are biosynthesised during the intraerythrocytic stages of Plasmodium falciparum.

Direct analysis of polyisoprenoid alcohols by electrospray ionization mass spectrometry (ESI-MS) often produces poor results requiring off-line time- and sample-consuming derivatization techniques. In this chapter, we describe a simple ESI-MS approach for the direct analysis of polyisoprenoid alcohols from biological samples. Lithium iodide is used to promote cationization by intense formation of [M+Li](+) adducts. Detection of polyisoprenoids with mass determination can thus be performed with high sensitivity (LOD near 100 pM), whereas characteristic collision-induced dissociations observed for both dolichols and polyprenols permit investigation of their structure. We also describe a simple ESI-MS approach for the direct analysis of carotenoids in biological samples using lithium iodide to promote their ionization and the analysis of several carotenoids as proof-of-principle cases. Finally, we applied ESI(Li(+))-MS and ESI(Li(+))-MS/MS to investigate the presence of carotenoids in Plasmodium falciparum.

Carotenoids are widespread lipophilic pigments synthesized by all photosynthetic organisms and some nonphotosynthetic fungi and bacteria. All carotenoids are derived from the C40 isoprenoid precursor geranylgeranyl pyrophosphate, and their chemical and physical properties are associated with light absorption, free radical scavenging, and antioxidant activity. Carotenoids are generally synthesized in well defined subcellular organelles, the plastids, which are also present in the phylum Apicomplexa, which comprises a number of important human parasites, such as Plasmodium and Toxoplasma. Recently, it was demonstrated that Toxoplasma gondii synthesizes abscisic acid. We therefore asked if Plasmodium falciparum is also capable of synthesizing carotenoids. Herein, biochemical findings demonstrated the presence of carotenoid biosynthesis in the intraerythrocytic stages of the apicomplexan parasite P. falciparum. Using metabolic labeling with radioisotopes, in vitro inhibition tests with norflurazon, a specific inhibitor of plant carotenoid biosynthesis, the results showed that intraerythrocytic stages of P. falciparum synthesize carotenoid compounds. A plasmodial enzyme that presented phytoene synthase activity was also identified and characterized. These findings not only contribute to the current understanding of P. falciparum evolution but shed light on a pathway that could serve as a chemotherapeutic target.

Direct analysis of polyisoprenoid alcohols by electrospray ionization mass spectrometry (ESI-MS) often produces poor results requiring off-line time- and sample-consuming derivatization techniques. In this chapter, we describe a simple ESI-MS approach for the direct analysis of polyisoprenoid alcohols from biological samples. Lithium iodide is used to promote cationization by intense formation of [M+Li](+) adducts. Detection of polyisoprenoids with mass determination can thus be performed with high sensitivity (LOD near 100 pM), whereas characteristic collision-induced dissociations observed for both dolichols and polyprenols permit investigation of their structure. We also describe a simple ESI-MS approach for the direct analysis of carotenoids in biological samples using lithium iodide to promote their ionization and the analysis of several carotenoids as proof-of-principle cases. Finally, we applied ESI(Li(+))-MS and ESI(Li(+))-MS/MS to investigate the presence of carotenoids in Plasmodium falciparum.

Using an antigen-capture, dot-blot assay, antigens were detected in the urine of 50 patients infected with Plasmodium falciparum. Antigens were also detected in 12/15 patients who had no detectable parasitemias 1–2 weeks after chemotherapy. By Western blotting and immunoprecipitation, four predominant antigens were identified with the following molecular masses (Mr) and isoelectric points (pI): antigen 1, 200 kDa, pI 6.4–6.27; antigen 2, 180 kDa, pI 5.2–4.8; antigen 3, 150 kDa, pI 5.5; antigen 4, 96 kDa, pI 5.1–4.8. These antigens were heat stable to 100°C for 5 min. Antigens were also detected in the urine of 35 patients with acute P. vivax infections by Western blotting and dot-blot analysis and 10/10 patients three weeks following chemotherapy. The antigens had Mr of 200, 170, and 130 kDa.

Development of new drugs is one of the strategies for malaria control. The biosynthesis of several isoprenoids inPlasmodium falciparumwas recently described. Interestingly, some intermediates and final products biosynthesized by this pathway in mammals differ from those biosynthesized inP. falciparum. These facts prompted us to evaluate various terpenes, molecules with a similar chemical structure to the intermediates of the isoprenoids pathway, as potential antimalarial drugs. Different terpenes andS-farnesylthiosalicylic acid were tested on cultures of the intraerythrocytic stages ofP. falciparum, and the 50% inhibitory concentrations for each one were found: farnesol, 64 μM; nerolidol, 760 nM; limonene, 1.22 mM; linalool, 0.28 mM; andS-farnesylthiosalicylic acid, 14 μM. All the terpenes tested inhibited dolichol biosynthesis in the trophozoite and schizont stages when [1-(n)-3H]farnesyl pyrophosphate triammonium salt ([3H]FPP) was used as precursor. Farnesol, nerolidol, and linalool showed stronger inhibitory activity on the biosynthesis of the isoprenic side chain of the benzoquinone ring of ubiquinones in the schizont stage. Treatment of schizont stages withS-farnesylthiosalicylic acid led to a decrease in intensity of the band corresponding a p21rasprotein. The inhibitory effect of terpenes andS-farnesylthiosalicylic acid on the biosynthesis of both dolichol and the isoprenic side chain of ubiquinones and the isoprenylation of proteins in the intraerythrocytic stages ofP. falciparumappears to be specific, because overall protein biosynthesis was not affected. Combinations of some terpenes orS-farnesylthiosalicylic acid tested in this work with other antimalarial drugs, like fosmidomycin, could be a new strategy for the treatment of malaria.

Isoprenylation is an essential protein modification in eukaryotic cells. Herein, we report that in Plasmodium falciparum , a number of proteins were labeled upon incubation of intraerythrocytic forms with either [ 3 H]farnesyl pyrophosphate or [ 3 H]geranylgeranyl pyrophosphate. By thin-layer chromatography, we showed that attached isoprenoids are partially modified to dolichol and other, uncharacterized, residues, confirming active isoprenoid metabolism in this parasite. Incubation of blood-stage P. falciparum treated with the isoprenylation inhibitor limonene significantly decreased the parasites' progression from the ring stage to the trophozoite stage and at 1.22 mM, 50% of the parasites died after the first cycle. Using Ras- and Rap-specific monoclonal antibodies, putative Rap and Ras proteins of P. falciparum were immunoprecipitated. Upon treatment with 0.5 mM limonene, isoprenylation of these proteins was significantly decreased, possibly explaining the observed arrest of parasite development.

The 2-C-methyl-d-erythritol-4-phosphate and shikimate pathways were found to be active in Plasmodium falciparum and both can result in vitamin E biosynthesis in plants and algae. This study biochemically confirmed vitamin E biosynthesis in the malaria parasite, which can be inhibited by usnic acid. Furthermore, we found evidence pointing to a role of this vitamin in infected erythrocytes. These findings not only contribute to current understanding of P. falciparum biology but also reveal a pathway that could serve as a chemotherapeutic target.

Herein, we show that intraerythrocytic stages of Plasmodium falciparum have an active pathway for biosynthesis of menaquinone. Kinetic assays confirmed that plasmodial menaquinone acts at least in the electron transport. Similarly to Escherichia coli, we observed increased levels of menaquinone in parasites kept under anaerobic conditions. Additionally, the mycobacterial inhibitor of menaquinone synthesis Ro 48-8071 also suppressed menaquinone biosynthesis and growth of parasites, although off-targets may play a role in this growth-inhibitory effect. Due to its absence in humans, the menaquinone biosynthesis can be considered an important drug target for malaria.