pyrophosphate and Toxoplasmosis

Many biosynthetic pathways produce pyrophosphate (PPi) as a by-product, which is cytotoxic if accumulated at high levels. Pyrophosphatases play pivotal roles in PPi detoxification by converting PPi to inorganic phosphate. A number of apicomplexan parasites, including Toxoplasma gondii and Cryptosporidium parvum, express a PPi-dependent phosphofructokinase (PPi-PFK) that consumes PPi to power the phosphorylation of fructose-6-phosphate. However, the physiological roles of PPi-PFKs in these organisms are not known. Here, we report that Toxoplasma expresses both ATP- and PPi-dependent phosphofructokinases in the cytoplasm. Nonetheless, only PPi-PFK was indispensable for parasite growth, whereas the deletion of ATP-PFK did not affect parasite proliferation or virulence. The conditional depletion of PPi-PFK completely arrested parasite growth, but it did not affect the ATP level and only modestly reduced the flux of central carbon metabolism. However, PPi-PFK depletion caused a significant increase in cellular PPi and decreased the rates of nascent protein synthesis. The expression of a cytosolic pyrophosphatase in the PPi-PFK depletion mutant reduced its PPi level and increased the protein synthesis rate, therefore partially rescuing its growth. These results suggest that PPi-PFK has a major role in maintaining pyrophosphate homeostasis in T. gondii. This role may allow PPi-PFK to fine-tune the balance of catabolism and anabolism and maximize the utilization efficiency for carbon nutrients derived from host cells, increasing the success of parasitism. Moreover, PPi-PFK is essential for parasite propagation and virulence in vivo but it is not present in human hosts, making it a potential drug target to combat toxoplasmosis.

Topics: Adenosine Triphosphate; Carbohydrate Metabolism; Diphosphates; Homeostasis; Mutation; Phosphorylation; Phosphotransferases; Toxoplasma; Toxoplasmosis

Prenyldiphosphate synthases catalyze the reaction of allylic diphosphates with one or more isopentenyl diphosphate molecules to form compounds such as farnesyl diphosphate, used in, e.g., sterol biosynthesis and protein prenylation, as well as longer "polyprenyl" diphosphates, used in ubiquinone and menaquinone biosynthesis. Quinones play an essential role in electron transport and are associated with the inner mitochondrial membrane due to the presence of the polyprenyl group. In this work, we investigated the synthesis of the polyprenyl diphosphate that alkylates the ubiquinone ring precursor in Toxoplasma gondii, an opportunistic pathogen that causes serious disease in immunocompromised patients and the unborn fetus. The enzyme that catalyzes this early step of the ubiquinone synthesis is Coq1 (TgCoq1), and we show that it produces the C35 species heptaprenyl diphosphate. TgCoq1 localizes to the mitochondrion and is essential for

Topics: Animals; Diphosphates; Diphosphonates; Mice; Sterols; Toxoplasma; Toxoplasmosis; Ubiquinone; Vitamin K 2

Intracellular pathogens have complex metabolic interactions with their host cells to ensure a steady supply of energy and anabolic building blocks for rapid growth. Here we use the obligate intracellular parasite Toxoplasma gondii to probe this interaction for isoprenoids, abundant lipidic compounds essential to many cellular processes including signaling, trafficking, energy metabolism, and protein translation. Synthesis of precursors for isoprenoids in Apicomplexa occurs in the apicoplast and is essential. To synthesize longer isoprenoids from these precursors, T. gondii expresses a bifunctional farnesyl diphosphate/geranylgeranyl diphosphate synthase (TgFPPS). In this work we construct and characterize T. gondii null mutants for this enzyme. Surprisingly, these mutants have only a mild growth phenotype and an isoprenoid composition similar to wild type parasites. However, when extracellular, the loss of the enzyme becomes phenotypically apparent. This strongly suggests that intracellular parasite salvage FPP and/or geranylgeranyl diphosphate (GGPP) from the host. We test this hypothesis using inhibitors of host cell isoprenoid synthesis. Mammals use the mevalonate pathway, which is susceptible to statins. We document strong synergy between statin treatment and pharmacological or genetic interference with the parasite isoprenoid pathway. Mice can be cured with atorvastatin (Lipitor) from a lethal infection with the TgFPPs mutant. We propose a double-hit strategy combining inhibitors of host and parasite pathways as a novel therapeutic approach against Apicomplexan parasites.

Topics: Animals; Apicoplasts; Atorvastatin; Diphosphates; Diterpenes; Farnesyltranstransferase; Female; Gene Knockdown Techniques; Heptanoic Acids; Humans; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Mice; Mice, Inbred BALB C; Polyisoprenyl Phosphates; Protozoan Proteins; Pyrroles; Sesquiterpenes; Toxoplasma; Toxoplasmosis

Farnesyl-diphosphate synthase (FPPS) catalyzes the synthesis of farnesyl diphosphate, an important precursor of sterols, dolichols, ubiquinones, and prenylated proteins. We report the cloning and characterization of two Toxoplasma gondii farnesyl-diphosphate synthase (TgFPPS) homologs. A single genetic locus produces two transcripts, TgFPPS and TgFPPSi, by alternative splicing. Both isoforms were heterologously expressed in Escherichia coli, but only TgFPPS was active. The protein products predicted from the nucleotide sequences have 646 and 605 amino acids and apparent molecular masses of 69.5 and 64.5 kDa, respectively. Several conserved sequence motifs found in other prenyl-diphosphate synthases are present in both TgFPPSs. TgFPPS was also expressed in the baculovirus system and was biochemically characterized. In contrast to the FPPS of other eukaryotic organisms, TgFPPS is bifunctional, catalyzing the formation of both farnesyl diphosphate and geranylgeranyl diphosphate. TgFPPS localizes to the mitochondria, as determined by the co-localisation of the affinity-purified antibodies against the protein with MitoTracker, and in accord with the presence of an N-terminal mitochondria-targeting signal in the protein. This enzyme is an attractive target for drug development, because the order of inhibition of the enzyme by a number of bisphosphonates is the same as that for inhibition of parasite growth. In summary, we report the first bifunctional farnesyl-diphosphate/geranylgeranyl-diphosphate synthase identified in eukaryotes, which, together with previous results, establishes this enzyme as a valid target for the chemotherapy of toxoplasmosis.

Topics: Alternative Splicing; Amino Acid Motifs; Amino Acid Sequence; Animals; Baculoviridae; Bone Density Conservation Agents; Catalysis; Cloning, Molecular; Diphosphates; Diphosphonates; Diterpenes; Dolichols; Drug Design; Enzyme Inhibitors; Escherichia coli; Farnesyltranstransferase; Gene Expression; Gene Expression Regulation, Enzymologic; Geranyltranstransferase; Isoenzymes; Molecular Sequence Data; Polyisoprenyl Phosphates; Protein Prenylation; Protozoan Proteins; Quantitative Trait Loci; Recombinant Proteins; Sesquiterpenes; Sterols; Toxoplasma; Toxoplasmosis; Ubiquinone