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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. Author summary: Falciparum malaria is a potentially fatal disease caused by the parasite Plasmodium falciparum. Antimalarials such as fosmidomycin and clindamycin target a critical pathway in the parasite, crucial for producing certain substances essential for the parasite's survival, particularly phosphorylated isoprenoids. However, the limited effectiveness of these drugs in clinical trials for malaria treatment underscores the need for further related studies. Previous in vitro experiments have demonstrated that the parasite can utilize unphosphorylated isoprenoids, namely farnesol and geranylgeraniol, if they are present in the external environment. Thus, these substances act as antidotes, rendering the parasite resistant to both fosmidomycin and clindamycin. This study reveals for the first time that geranylgeraniol naturally occurs in the human body. Additionally, we have identified a novel enzyme, prenol kinase, which enables the parasite to use these unphosphorylated isoprenoids by converting them into their metabolically active phosphorylated counterparts. Parasites lacking the prenol kinase gene remain viable but become more susceptible to the effects of fosmidomycin, even in the presence of farnesol or geranylgeraniol. These findings suggest that the scavenging of unphosphorylated isoprenoids by the parasite might supplement its isoprenoid needs when the endogenous production is inhibited by drugs like fosmidomycin or clindamycin. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Farnesol salvage, a two-step pathway converting farnesol to farnesyl pyrophosphate (FPP), occurs in bacteria, plants, and animals. This paper investigates the presence of this pathway in fungi. Through bioinformatics, biochemistry, and physiological analyses, we demonstrate its absence in the yeasts Saccharomyces cerevisiae and Candida albicans, suggesting a likely absence across fungi. We screened 1,053 fungal genomes, including 34 from C. albicans, for potential homologs to four genes (Arabidopsis thaliana AtFOLK, AtVTE5, AtVTE6, and Plasmodium falciparum PfPOLK) known to accomplish farnesol/prenol salvage in other organisms. Additionally, we showed that 3H-farnesol was not converted to FPP or any other phosphorylated prenol, and exogenous farnesol was not metabolized within 90 minutes at any phase of growth and did not rescue cells from the toxic effects of atorvastatin, but it did elevate the levels of intracellular farnesol (Fi). All these experiments were conducted with C. albicans. In sum, we found no evidence for farnesol salvage in fungi. [ABSTRACT FROM AUTHOR]