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.

Presence of Phylloquinone in the Intraerythrocytic Stages of Plasmodium falciparum.

Malaria is one of the most widespread parasitic diseases, especially in Africa, Southeast Asia and South America. One of the greatest problems for control of the disease is the emergence of drug resistance, which leads to a need for the development of new antimalarial compounds. The biosynthesis of isoprenoids has been investigated as part of a strategy to identify new targets to obtain new antimalarial drugs. Several isoprenoid quinones, including menaquinone-4 (MK-4/vitamin K2), α- and γ-tocopherol and ubiquinone (UQ) homologs UQ-8 and UQ-9, were previously detected in in vitro cultures of Plasmodium falciparum in asexual stages. Herein, we described for the first time the presence of phylloquinone (PK/vitamin K1) in P. falciparum and discuss the possible origins of this prenylquinone. While our results in metabolic labeling experiments suggest a biosynthesis of PK prenylation via phytyl pyrophosphate (phytyl-PP) with phytol being phosphorylated, on the other hand, exogenous PK attenuated atovaquone effects on parasitic growth and respiration, showing that this metabolite can be transported from extracellular environment and that the mitochondrial electron transport system (ETS) of P. falciparum is capable to interact with PK. Although the natural role and origin of PK remains elusive, this work highlights the PK importance in plasmodial metabolism and future studies will be important to elucidate in seeking new targets for antimalarial drugs.

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.

Glutamine Analogues Impair Cell Proliferation, the Intracellular Cycle and Metacyclogenesis in Trypanosoma cruzi.

Trypanosoma cruzi is the aetiologic agent of Chagas disease, which affects people in the Americas and worldwide. The parasite has a complex life cycle that alternates among mammalian hosts and insect vectors. During its life cycle, T. cruzi passes through different environments and faces nutrient shortages. It has been established that amino acids, such as proline, histidine, alanine, and glutamate, are crucial to T. cruzi survival. Recently, we described that T. cruzi can biosynthesize glutamine from glutamate and/or obtain it from the extracellular environment, and the role of glutamine in energetic metabolism and metacyclogenesis was demonstrated. In this study, we analysed the effect of glutamine analogues on the parasite life cycle. Here, we show that glutamine analogues impair cell proliferation, the developmental cycle during the infection of mammalian host cells and metacyclogenesis. Taken together, these results show that glutamine is an important metabolite for T. cruzi survival and suggest that glutamine analogues can be used as scaffolds for the development of new trypanocidal drugs. These data also reinforce the supposition that glutamine metabolism is an unexplored possible therapeutic target.

A Fluorinated Phenylbenzothiazole Arrests the Trypanosoma cruzi Cell Cycle and Diminishes the Infection of Mammalian Host Cells.

Chagas disease (CD) is a human infection caused by Trypanosoma cruzi CD was traditionally endemic to the Americas; however, due to migration it has spread to countries where it is not endemic. The current chemotherapy to treat CD induces several side effects, and its effectiveness in the chronic phase of the disease is controversial. In this contribution, substituted phenylbenzothiazole derivatives were synthesized and biologically evaluated as trypanocidal agents against Trypanosoma cruzi The trypanocidal activities of the most promising compounds were determined through systematic in vitro screening, and their modes of action were determined as well. The physicochemical-structural characteristics responsible for the trypanocidal effects were identified, and their possible therapeutic application in Chagas disease is discussed. Our results show that the fluorinated compound 2-methoxy-4-[5-(trifluoromethyl)-1,3-benzothiazol-2-yl] phenol (BT10) has the ability to inhibit the proliferation of epimastigotes [IC50(Epi) = 23.1 ± 1.75 µM] and intracellular forms of trypomastigotes [IC50(Tryp) = 8.5 ± 2.9 µM] and diminishes the infection index by more than 80%. In addition, BT10 has the ability to selectively fragment 68% of the kinetoplastid DNA compared with 5% of nucleus DNA. The mode of action for BT10 on T. cruzi suggests that the development of fluorinated phenylbenzothiazole with electron-withdrawing substituent is a promising strategy for the design of trypanocidal drugs.

A Fluorinated phenylbenzothiazole arrests the trypanosoma cruzi cell cycle and diminishes the Infection of mammalian host cells

Chagas disease (CD) is a human infection caused by Trypanosoma cruzi. CD was traditionally endemic to the Americas; however, due to migration it has spread to countries where it is not endemic. The current chemotherapy to treat CD induces several side effects, and its effectiveness in the chronic phase of the disease is controversial. In this contribution, substituted phenylbenzothiazole derivatives were synthesized and biologically evaluated as trypanocidal agents against Trypanosoma cruzi. The trypanocidal activities of the most promising compounds were determined through systematic in vitro screening, and their modes of action were determined as well. The physicochemical-structural characteristics responsible for the trypanocidal effects were identified, and their possible therapeutic application in Chagas disease is discussed. Our results show that the fluorinated compound 2-methoxy-4-[5-(trifluoromethyl)-1,3-benzothiazol-2-yl] phenol (BT10) has the ability to inhibit the proliferation of epimastigotes [IC50(Epi) = 23.1 ± 1.75 µM] and intracellular forms of trypomastigotes [IC50(Tryp) = 8.5 ± 2.9 µM] and diminishes the infection index by more than 80%. In addition, BT10 has the ability to selectively fragment 68% of the kinetoplastid DNA compared with 5% of nucleus DNA. The mode of action for BT10 on T. cruzi suggests that the development of fluorinated phenylbenzothiazole with electron-withdrawing substituent is a promising strategy for the design of trypanocidal drugs.

Prenylquinones in Human Parasitic Protozoa: Biosynthesis, Physiological Functions, and Potential as Chemotherapeutic Targets.

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.

Uptake of l-Alanine and Its Distinct Roles in the Bioenergetics of Trypanosoma cruzi.

Amino acids participate in several critical processes in the biology of trypanosomatids, such as osmoregulation, cell differentiation, and host cell invasion. Some of them provide reducing power for mitochondrial ATP synthesis. It was previously shown that alanine, which is formed mainly by the amination of pyruvate, is a metabolic end product formed when parasites are replicating in a medium rich in glucose and amino acids. It was shown as well that this amino acid can also be used for the regulation of cell volume and resistance to osmotic stress. In this work, we demonstrate that, despite it being an end product of its metabolism, Trypanosoma cruzi can take up and metabolize l-Ala through a low-specificity nonstereoselective active transport system. The uptake was dependent on the temperature in the range between 10 and 40°C, which allowed us to calculate an activation energy of 66.4 kJ/mol and estimate the number of transporters per cell at ~436,000. We show as well that, once taken up by the cells, l-Ala can be completely oxidized to CO2, supplying electrons to the electron transport chain, maintaining the electrochemical proton gradient across the mitochondrial inner membrane, and supporting ATP synthesis in T. cruzi epimastigotes. Our data demonstrate a dual role for Ala in the parasite's bioenergetics, by being a secreted end product of glucose catabolism and taken up as nutrient for oxidative mitochondrial metabolism.IMPORTANCE It is well known that trypanosomatids such as the etiological agent of Chagas' disease, Trypanosoma cruzi, produce alanine as a main end product of their energy metabolism when they grow in a medium containing glucose and amino acids. In this work, we investigated if under starvation conditions (which happen during the parasite life cycle) the secreted alanine could be recovered from the extracellular medium and used as an energy source. Herein we show that indeed, in parasites submitted to metabolic stress, this metabolite can be taken up and used as an energy source for ATP synthesis, allowing the parasite to extend its survival under starvation conditions. The obtained results point to a dual role for Ala in the parasite's bioenergetics, by being a secreted end product of glucose catabolism and taken up as nutrient for oxidative mitochondrial metabolism.

The glutamine synthetase of Trypanosoma cruzi is required for its resistance to ammonium accumulation and evasion of the parasitophorous vacuole during host-cell infection.

Trypanosoma cruzi, the etiological agent of Chagas disease, consumes glucose and amino acids depending on the environmental availability of each nutrient during its complex life cycle. For example, amino acids are the major energy and carbon sources in the intracellular stages of the T. cruzi parasite, but their consumption produces an accumulation of NH4+ in the environment, which is toxic. These parasites do not have a functional urea cycle to secrete excess nitrogen as low-toxicity waste. Glutamine synthetase (GS) plays a central role in regulating the carbon/nitrogen balance in the metabolism of most living organisms. We show here that the gene TcGS from T. cruzi encodes a functional glutamine synthetase; it can complement a defect in the GLN1 gene from Saccharomyces cerevisiae and utilizes ATP, glutamate and ammonium to yield glutamine in vitro. Overall, its kinetic characteristics are similar to other eukaryotic enzymes, and it is dependent on divalent cations. Its cytosolic/mitochondrial localization was confirmed by immunofluorescence. Inhibition by Methionine sulfoximine revealed that GS activity is indispensable under excess ammonium conditions. Coincidently, its expression levels are maximal in the amastigote stage of the life cycle, when amino acids are preferably consumed, and NH4+ production is predictable. During host-cell invasion, TcGS is required for the parasite to escape from the parasitophorous vacuole, a process sine qua non for the parasite to replicate and establish infection in host cells. These results are the first to establish a link between the activity of a metabolic enzyme and the ability of a parasite to reach its intracellular niche to replicate and establish host-cell infection.

The glutamine synthetase of Trypanosoma cruzi is required for its resistance to ammonium accumulation and evasion of the parasitophorous vacuole during host-cell infection

Trypanosoma cruzi, the etiological agent of Chagas disease, consumes glucose and amino acids depending on the environmental availability of each nutrient during its complex life cycle. For example, amino acids are the major energy and carbon sources in the intracellular stages of the T. cruzi parasite, but their consumption produces an accumulation of NH4+ in the environment, which is toxic. These parasites do not have a functional urea cycle to secrete excess nitrogen as low-toxicity waste. Glutamine synthetase (GS) plays a central role in regulating the carbon/nitrogen balance in the metabolism of most living organisms. We show here that the gene TcGS from T. cruzi encodes a functional glutamine synthetase; it can complement a defect in the GLN1 gene from Saccharomyces cerevisiae and utilizes ATP, glutamate and ammonium to yield glutamine in vitro. Overall, its kinetic characteristics are similar to other eukaryotic enzymes, and it is dependent on divalent cations. Its cytosolic/ mitochondrial localization was confirmed by immunofluorescence. Inhibition by Methionine sulfoximine revealed that GS activity is indispensable under excess ammonium conditions. Coincidently, its expression levels are maximal in the amastigote stage of the life cycle, when amino acids are preferably consumed, and NH4+ production is predictable. During host-cell invasion, TcGS is required for the parasite to escape from the parasitophorous vacuole, a process sine qua non for the parasite to replicate and establish infection in host cells. These results are the first to establish a link between the activity of a metabolic enzyme and the ability of a parasite to reach its intracellular niche to replicate and establish host-cell infection.

An Aromatic Diamidine That Targets Kinetoplast DNA, Impairs the Cell Cycle in Trypanosoma cruzi, and Diminishes Trypomastigote Release from Infected Mammalian Host Cells.

Trypanosoma cruzi is the etiological agent of Chagas disease, affecting approximately 10 million people in the Americas and with some 40 million people at risk. The objective of this study was to evaluate the anti-T. cruzi activity of three new diamidines that have a 3,4-ethylenedioxy extension of the thiophene core, designated MB17, MB19, and MB38. All three diamidines exhibited dose-dependent inhibition of epimastigote replication. The mechanisms of action of these diamidines were investigated. Unlike MB17 and MB19, MB38 exhibited a significant increase in the number of annexin-propidium iodide double-labeled cells compared to levels in control parasites. As MB17 had shown a lower 50% inhibitory concentration (IC50) against epimastigote growth, the mechanism of action of this drug was studied in more detail. MB17 triggered a decrease in the intracellular ATP levels. As a consequence, MB17 affected the genomic DNA and kinetoplast DNA (kDNA) and impaired the parasite cell cycle. Moreover, MB17 caused DNA fragmentation, with a more severe effect on kDNA than on nuclear DNA, resulting in dyskinetoplastic cells. MB17 was tested for toxicity and effectiveness for the treatment of infected CHO-K1 cells, exhibiting a 50% cytotoxic concentration (CC50) of 13.47 ± 0.37 µM and an IC50 of 0.14 ± 0.12 µM against trypomastigote release. MB17 also diminished the infection index by 60% at 0.5 µM. In conclusion, despite belonging to the same family, these diamidines have different efficiencies. To summarize, MB17 was the most potent of these diamidines against epimastigotes, producing DNA damage preferentially in kDNA, impairing the parasite cell cycle, and decreasing the infection index and trypomastigote release from infected mammalian host cells, with a high selectivity index (SI) (<90). These data suggest that MB17 could be an interesting lead compound against T. cruzi.