geranyl-pyrophosphate and Toxoplasmosis

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