Beyond the MEP Pathway: A novel kinase required for prenol utilization by malaria parasites.

A proposed treatment for malaria is a combination of fosmidomycin and clindamycin. Both compounds inhibit the methylerythritol 4-phosphate (MEP) pathway, the parasitic source of farnesyl and geranylgeranyl pyrophosphate (FPP and GGPP, respectively). Both FPP and GGPP are crucial for the biosynthesis of several essential metabolites such as ubiquinone and dolichol, as well as for protein prenylation. Dietary prenols, such as farnesol (FOH) and geranylgeraniol (GGOH), can rescue parasites from MEP inhibitors, suggesting the existence of a missing pathway for prenol salvage via phosphorylation. In this study, we identified a gene in the genome of P. falciparum, encoding a transmembrane prenol kinase (PolK) involved in the salvage of FOH and GGOH. The enzyme was expressed in Saccharomyces cerevisiae, and its FOH/GGOH kinase activities were experimentally validated. Furthermore, conditional knockout parasites (Δ-PolK) were created to investigate the biological importance of the FOH/GGOH salvage pathway. Δ-PolK parasites were viable but displayed increased susceptibility to fosmidomycin. Their sensitivity to MEP inhibitors could not be rescued by adding prenols. Additionally, Δ-PolK parasites lost their capability to utilize prenols for protein prenylation. Experiments using culture medium supplemented with whole/delipidated human plasma in transgenic parasites revealed that human plasma has components that can diminish the effectiveness of fosmidomycin. Mass spectrometry tests indicated that both bovine supplements used in culture and human plasma contain GGOH. These findings suggest that the FOH/GGOH salvage pathway might offer an alternate source of isoprenoids for malaria parasites when de novo biosynthesis is inhibited. This study also identifies a novel kind of enzyme related to isoprenoid metabolism.

Plasmodium falciparum COQ2 gene encodes a functional 4-hydroxybenzoate polyprenyltransferase.

Ubiquinone (UQ) is a fundamental mitochondrial electron transport chain component. This compound is synthesized as the condensation of a p-substituted benzoic acid and a polyisoprenic moiety catalyzed by the enzyme 4-hydroxybenzoate polyprenyltransferase (EC 2.5.1.39). In Plasmodium spp., this enzyme is still uncharacterized. In this work, we expressed the sequence of the Plasmodium falciparum PF3D7_0607500 gene (abbreviated as PfCOQ2) in a coq2Δ mutant strain of Saccharomyces cerevisiae, and studied the functionality of its gene product. This open reading frame could complement S. cerevisiae coq2Δ mutant growth defect on media with glycerol as a carbon source. Further, UQ was unequivocally identified in lipid extracts from this coq2Δ mutant when expressing PfCOQ2. Remarkably, UQ was detected under those conditions when S. cerevisiae cells were metabolically labeled with either [ring-14C(U)]-p-aminobenzoic acid or [ring-14C(U)]-4-hydroxybenzoic acid. However, no UQ was detected in P. falciparum if labeled with p-aminobenzoic acid. These results indicate that PfCOQ2 is a 4-hydroxybenzoate polyprenyltransferase. Further, its substrate profile seems not dissimilar to that of S. cerevisiae, but, as in other organisms, p-aminobenzoic acid does not act as an aromatic precursor in UQ biosynthesis in P. falciparum. The reason for this last feature remains to be established, but may lie upstream of PfCOQ2.

The Biomedical Importance of the Missing Pathway for Farnesol and Geranylgeraniol Salvage.

Isoprenoids are the output of the polymerization of five-carbon, branched isoprenic chains derived from isopentenyl pyrophosphate (IPP) and its isomer, dimethylallyl pyrophosphate (DMAPP). Isoprene units are consecutively condensed to form longer structures such as farnesyl and geranylgeranyl pyrophosphate (FPP and GGPP, respectively), necessary for the biosynthesis of several metabolites. Polyprenyl transferases and synthases use polyprenyl pyrophosphates as their natural substrates; however, it is known that free polyprenols, such as farnesol (FOH), and geranylgeraniol (GGOH) can be incorporated into prenylated proteins, ubiquinone, cholesterol, and dolichols. Furthermore, FOH and GGOH have been shown to block the effects of isoprenoid biosynthesis inhibitors such as fosmidomycin, bisphosphonates, or statins in several organisms. This phenomenon is the consequence of a short pathway, which was observed for the first time more than 25 years ago: the polyprenol salvage pathway, which works via the phosphorylation of FOH and GGOH. Biochemical studies in bacteria, animals, and plants suggest that this pathway can be carried out by two enzymes: a polyprenol kinase and a polyprenyl-phosphate kinase. However, to date, only a few genes have been unequivocally identified to encode these enzymes in photosynthetic organisms. Nevertheless, pieces of evidence for the importance of this pathway abound in studies related to infectious diseases, cancer, dyslipidemias, and nutrition, and to the mitigation of the secondary effects of several drugs. Furthermore, nowadays it is known that both FOH and GGOH can be incorporated via dietary sources that produce various biological effects. This review presents, in a simplified but comprehensive manner, the most important data on the FOH and GGOH salvage pathway, stressing its biomedical importance The main objective of this review is to bring to light the need to discover and characterize the kinases associated with the isoprenoid salvage pathway in animals and pathogens.

Isoprenoid alcohols utilization by malaria parasites.

Plasmodium falciparum is the etiological agent of human malaria, one of the most widespread diseases in tropical and subtropical regions. Drug resistance is one of the biggest problems in controlling the disease, which leads to the need to discover new antimalarial compounds. One of the most promissory drugs purposed is fosmidomycin, an inhibitor of the biosynthesis of isoprene units by the methylerythritol 4-phosphate (MEP) pathway, which in some cases failed in clinical studies. Once formed, isoprene units are condensed to form longer structures such as farnesyl and geranylgeranyl pyrophosphate, which are necessary for Heme O and A formation, ubiquinone, and dolichyl phosphate biosynthesis as well as for protein isoprenylation. Even though the natural substrates of polyprenyl transferases and synthases are polyprenyl pyrophosphates, it was already demonstrated that isoprenoid alcohols (polyprenols) such as farnesol (FOH) and geranylgeraniol (GGOH) can rescue parasites from fosmidomycin. This study better investigated how this rescue phenomenon occurs by performing drug-rescue assays. Similarly, to FOH and GGOH, it was observed that phytol (POH), a 20-carbon plant isoprenoid, as well as unsaponifiable lipid extracts from foods rescue parasites from the antimalarial effect of fosmidomycin. Contrarily, neither dolichols nor nonaprenol rescue parasites from fosmidomycin. Considering this, here we characterized the transport of FOH, GGOH, and POH. Once incorporated, it was observed that these substances are phosphorylated, condensed into longer isoprenoid alcohols, and incorporated into proteins and dolichyl phosphates. Through proteomic and radiolabelling approaches, it was found that prenylated proteins are naturally attached to several isoprenoids, derived from GGOH, dolichol, and POH if exogenously added. Furthermore, the results suggest the presence of at least two promiscuous protein prenyltransferases in the parasite: one enzyme which can use FPP among other unidentified substrates and another enzyme that can use GGPP, phytyl pyrophosphate (PPP), and dolichols, among other substrates not identified here. Thus, further evidence was obtained for dolichols and other isoprenoid products attached to proteins. This study helps to better understand the apicoplast-targeting antimalarial mechanism of action and a novel post-translational modification of proteins in P. falciparum.

Suitability of methods for Plasmodium falciparum cultivation in atmospheric air.

BACKGROUND: One of the most controversial factors about malaria parasite culture is the gaseous composition used. The most commonly used one consists of a mixture poor in O2 and rich in CO2. OBJECTIVES: The present study aimed to share standard methods from our research group simplifying Plasmodium falciparum cultures by employing atmospheric air (ATM) and reusable glass bottles under agitation. METHODS: Here, it was compared the parasite viability, free oxygen in media, and drug sensitivity between different strains and isolates maintained for long periods under ATM or classic conditions. FINDINGS: The oxygen concentration in media under ATM was slightly superior to that observed in human blood and the media under the classic gaseous mixture. However, ATM or the use of glass bottles did not affect parasitic proliferation after several years of culture. Noticeably, the introduction of ATM altered reversibly the efficacy of several antimalarials. This influence was different between the strains and isolate. CONCLUSIONS: ATM conditions and shaken flasks could be used as a standard method condition for culture manutention since they do not differ greatly from classical 5% O2 gas mixtures in terms of parasite proliferation and do not impose non-reversible changes to P. falciparum physiology.

Suitability of methods for Plasmodium falciparum cultivation in atmospheric air

BACKGROUND One of the most controversial factors about malaria parasite culture is the gaseous composition used. The most commonly used one consists of a mixture poor in O2 and rich in CO2. OBJECTIVES The present study aimed to share standard methods from our research group simplifying Plasmodium falciparum cultures by employing atmospheric air (ATM) and reusable glass bottles under agitation. METHODS Here, it was compared the parasite viability, free oxygen in media, and drug sensitivity between different strains and isolates maintained for long periods under ATM or classic conditions. FINDINGS The oxygen concentration in media under ATM was slightly superior to that observed in human blood and the media under the classic gaseous mixture. However, ATM or the use of glass bottles did not affect parasitic proliferation after several years of culture. Noticeably, the introduction of ATM altered reversibly the efficacy of several antimalarials. This influence was different between the strains and isolate. CONCLUSIONS ATM conditions and shaken flasks could be used as a standard method condition for culture manutention since they do not differ greatly from classical 5% O2 gas mixtures in terms of parasite proliferation and do not impose non-reversible changes to P. falciparum physiology.

Quantification of nerolidol in mouse plasma using gas chromatography-mass spectrometry.

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.

In vivo antimalarial activity and mechanisms of action of 4-nerolidylcatechol derivatives.

4-Nerolidylcatechol (1) is an abundant antiplasmodial metabolite that is isolated from Piper peltatum roots. O-Acylation or O-alkylation of compound 1 provides derivatives exhibiting improved stability and significant in vitro antiplasmodial activity. The aim of this work was to study the in vitro inhibition of hemozoin formation, inhibition of isoprenoid biosynthesis in Plasmodium falciparum cultures, and in vivo antimalarial activity of several 4-nerolidylcatechol derivatives. 1,2-O,O-Diacetyl-4-nerolidylcatechol (2) inhibited in vitro hemozoin formation by up to 50%. In metabolic labeling studies using [1-(n)-(3)H]geranylgeranyl pyrophosphate, diester 2: significantly inhibited the biosynthesis of isoprenoid metabolites ubiquinone 8, menaquinone 4, and dolichol 12 in cultures of P. falciparum 3D7. Similarly, 2-O-benzyl-4-nerolidylcatechol (3) significantly inhibited the biosynthesis of dolichol 12. P. falciparum in vitro protein synthesis was not affected by compounds 2 or 3. At oral doses of 50 mg per kg of body weight per day, compound 2 suppressed Plasmodium berghei NK65 in infected BALB/c mice by 44%. This in vivo result for derivative 2 represents marked improvement over that obtained previously for natural product 1. Compound 2 was not detected in mouse blood 1 h after oral ingestion or in mixtures with mouse blood/blood plasma in vitro. However, it was detected after in vitro contact with human blood or blood plasma. Derivatives of 4-nerolidylcatechol exhibit parasite-specific modes of action, such as inhibition of isoprenoid biosynthesis and inhibition of hemozoin formation, and they therefore merit further investigation for their antimalarial potential.

Isoprenoid biosynthesis in the erythrocytic stages of Plasmodium falciparum.

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.

Isoprenoid biosynthesis in the erythrocytic stages of Plasmodium falciparum

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.

In vitro and in vivo antiplasmodial activities of risedronate and its interference with protein prenylation in Plasmodium falciparum.

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 [IC50] 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.

Mass spectrometry analysis of polyisoprenoids alcohols and carotenoids via ESI(Li(+))-MS/MS.

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.

Protein dolichylation in Plasmodium falciparum.

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.

Electrospray ionization mass spectrometry analysis of polyisoprenoid alcohols via Li+ cationization.

Direct analysis of polyisoprenoids by electrospray ionization mass spectrometry (ESI-MS) often produces poor results requiring off-line time and sample-consuming derivatization techniques. We describe a simple ESI-MS approach for the direct analysis of polyisoprenoids using several dolichols and polyprenols with different chain sizes as proof-of-principle cases. Lithium iodide is used to promote cationization by intense formation of [M+Li]+ adducts. Thus, detection of polyisoprenoids with mass determination can be performed with high sensitivity (limit of detection [LOD] approximately 100 rhoM), whereas characteristic collision-induced dissociations observed for both dolichols and polyprenols permit investigation of their structure. Using ESI(Li+)-MS and ESI(Li+)-MS/MS analysis, we screened for polyprenol products of an octaprenyl pyrophosphate synthase of Plasmodium falciparum and dolichols in a complex mixture of compounds produced by Leishmania amazonensis and P. falciparum.

Characterization of the isoprenoid chain of coenzyme Q in Plasmodium falciparum.

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.

The IgG-subclass distribution of naturally acquired antibodies to Plasmodium falciparum, in relation to malaria exposure and severity

A critical role has been proposed for the switch from non-cytophilic IgG2 to cytophilic antibodies of IgG1 and IgG3 subclasses observed in the humoral immune responses to Plasmodium falciparum of some Africans. These Africans have acquired clinically immunity naturally, after several years of exposure to holo-endemic malaria. In the present study, the possibility that life-long exposure to low levels of malarial endemicity may be associated with changes in the IgG-subclass composition of antibodies to P. falciparum was investigated in a native Amazonian community. The subjects were 138 malaria-exposed but non-infected Karitiana Indians. In a separate investigation, the concentrations of IgG-subclass antibodies in acutely ill patients with severe malaria (N = 22) were compared with those in age- and sex-matched controls who had uncomplicated malaria (N = 44). Plasma concentrations of IgG against a detergent-soluble extract of P. falciparum schizonts were measured by quantitative ELISA, using indirect standardization. Among the Karitiana, the concentrations of anti-parasite antibodies of all subclasses increased with age, and there was no correlation between age and the proportion of such antibodies which was cytophilic. The predominance of cytophilic IgG1 and non-cytophilic IgG2 antibodies in all age-groups of the Karitiana provides an example of an intermediate pattern of immune responses to P. falciparum which contrasts with those previously described in both clinically immune and non-immune populations. Although mean concentrations of cytophilic IgG1 against P. falciparum were significantly higher in the controls than in the patients with severe malaria, there were no significant differences in other IgG subclasses. Lack of exposure to malaria in the past was associated with disease severity (odds ratio = 4.75; 95% confidence interval = 1.31-17.42), and may explain, at least partially, the occurrence of defective, low-IgG1 antibody responses to P. falciparum in those subjects who had severe malaria.