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/geranylgeranyldiphosphate synthase identified in eukaryotes, which, together with previous results, establishes this enzyme as a valid target for the chemotherapy of toxoplasmosis.Toxoplasma gondii is a pathogenic protozoan parasite that infects a wide range of vertebrate hosts, including humans. T. gondii has been recognized as a major opportunistic pathogen of fetuses from recently infected mothers and of immunocompromised patients, i.e. those with AIDS. Toxoplasmic encephalitis is associated with high mortality and morbidity and is one of the most common opportunistic infections of the central nervous system in human immunodeficiency virus-infected patients (1). The chemotherapy for toxoplasmosis is not ideal especially for the AIDS patient because there is relapse of the infection if the treatment is halted because of intolerance of the patient to the side effects. Almost all the drugs in use have toxicity associated with their continued use. In addition, most of the recommended treatments do not have an effect on the slow growing bradyzoite forms.Isoprenoids are the most diverse and abundant compounds occurring in nature. Many types of isoprenoids (e.g. steroids, cholesterol, retinoids, carotenoids, ubiquinones, and prenyl groups bound to proteins) are essential components of the cellular machinery of all organisms because of their roles in a variety of biological processes. Despite their structural and functional variety, all isoprenoids derive from a common precursor, isopentenyl diphosphate (IPP), 2 and its isomer, dimethylallyl diphosphate (...

Summary The vacuolar proton pyrophosphatase (H+-PPase) of Toxoplasma gondii (TgVP1), a membrane proton pump, localizes to acidocalcisomes and a novel lysosome-like compartment termed plant-like vacuole (PLV) or vacuolar compartment (VAC). We report the characterization of a T. gondii null mutant for the TgVP1 gene. Propagation of these mutants decreased significantly because of deficient attachment and invasion of host cells, which correlated with deficient microneme secretion. Processing of cathepsin L (CPL) in these mutants was deficient only when the parasites were incubated in the presence of low concentrations of the vacuolar H+-ATPase (V-H+-ATPase) inhibitor bafilomycin A1, suggesting that either TgVP1 or the T. gondii V-H+-ATPase (TgVATPase) are sufficient to support CPL processing. The lack of TgVP1 did not affect processing of micronemal proteins, indicating that it does not contribute to proMIC maturations. The TgVP1 null mutants were more sensitive to extracellular conditions and were less virulent in mice. We demonstrate that T. gondii tachyzoites possess regulatory volume decrease capability during hypo-osmotic stress and this ability is impaired in TgVP1 null mutants implicating TgVP1 in osmoregulation. We hypothesize that osmoregulation is needed for host cell invasion and that TgVP1 plays a role during the normal lytic cycle of T. gondii.

Trypanosomatid parasites are the causative agents of many neglected tropical diseases and there is currently considerable interest in targeting endogenous sterol biosynthesis in these organisms as a route to the development of novel anti-infective drugs. Here, we report the first x-ray crystallographic structures of the enzyme squalene synthase (SQS) from a trypanosomatid parasite, Trypanosoma cruzi, the causative agent of Chagas disease. We obtained five structures of T. cruzi SQS and eight structures of human SQS with four classes of inhibitors: the substrate-analog S-thiolo-farnesyl diphosphate, the quinuclidines E5700 and ER119884, several lipophilic bisphosphonates, and the thiocyanate WC-9, with the structures of the two very potent quinuclidines suggesting strategies for selective inhibitor development. We also show that the lipophilic bisphosphonates have low nM activity against T. cruzi and inhibit endogenous sterol biosynthesis and that E5700 acts synergistically with the azole drug, posaconazole. The determination of the structures of trypanosomatid and human SQS enzymes with a diverse set of inhibitors active in cells provides insights into SQS inhibition, of interest in the context of the development of drugs against Chagas disease.

Apicomplexan infections cause substantial morbidity and mortality, worldwide. New, improved therapies are needed. Herein, we create a next generation anti-apicomplexan lead compound, JAG21, a tetrahydroquinolone, with increased sp3-character to improve parasite selectivity. Relative to other cytochrome b inhibitors, JAG21 has improved solubility and ADMET properties, without need for pro-drug. JAG21 significantly reduces Toxoplasma gondii tachyzoites and encysted bradyzoites in vitro, and in primary and established chronic murine infections. Moreover, JAG21 treatment leads to 100% survival. Further, JAG21 is efficacious against drug-resistant Plasmodium falciparum in vitro. Causal prophylaxis and radical cure are achieved after P. berghei sporozoite infection with oral administration of a single dose (2.5 mg/kg) or 3 days treatment at reduced dose (0.625 mg/kg/day), eliminating parasitemia, and leading to 100% survival. Enzymatic, binding, and co-crystallography/pharmacophore studies demonstrate selectivity for apicomplexan relative to mammalian enzymes. JAG21 has significant promise as a pre-clinical candidate for prevention, treatment, and cure of toxoplasmosis and malaria.

Background: Trypanosoma cruzi regulates gene expression by means of post-transcriptional mechanisms. Results: A 43-nt U-rich element was found in the 3Ј-UTR of a large number of mRNAs that are more abundant in intracellular amastigotes. Conclusion:The 43-nt U-rich element might be involved in the modulation of abundance and/or translation of transcripts in amastigotes. Significance: Results suggest the existence of stage-specific RNA regulons in T. cruzi.