Wolfgang Bohne

Carbohydrate Metabolism in the Toxoplasma gondii Apicoplast: Localization of Three Glycolytic Isoenzymes, the Single Pyruvate Dehydrogenase Complex, and a Plastid Phosphate Translocator

ABSTRACT Many apicomplexan parasites, such as Toxoplasma gondii and Plasmodium species, possess a nonphotosynthetic plastid, referred to as the apicoplast, which is essential for the parasites’ viability and displays characteristics similar to those of nongreen plastids in plants. In this study, we localized several key enzymes of the carbohydrate metabolism of T. gondii to either the apicoplast or the cytosol by engineering parasites which express epitope-tagged fusion proteins. The cytosol contains a complete set of enzymes for glycolysis, which should enable the parasite to metabolize imported glucose into pyruvate. All the glycolytic enzymes, from phosphofructokinase up to pyruvate kinase, are present in the T. gondii genome, as duplicates and isoforms of triose phosphate isomerase, phosphoglycerate kinase, and pyruvate kinase were found to localize to the apicoplast. The mRNA expression levels of all genes with glycolytic products were compared between tachyzoites and bradyzoites; however, a strict bradyzoite-specific expression pattern was observed only for enolase I. The T. gondii genome encodes a single pyruvate dehydrogenase complex, which was located in the apicoplast and absent in the mitochondrion, as shown by targeting of epitope-tagged fusion proteins and by immunolocalization of the native pyruvate dehydrogenase complex. The exchange of metabolites between the cytosol and the apicoplast is likely to be mediated by a phosphate translocator which was localized to the apicoplast. Based on these localization studies, a model is proposed that explains the supply of the apicoplast with ATP and the reduction power, as well as the exchange of metabolites between the cytosol and the apicoplast.

The toxoplasma apicoplast phosphate translocator links cytosolic and apicoplast metabolism and is essential for parasite survival.

Apicomplexa are unicellular eukaryotic pathogens that carry a vestigial algal endosymbiont, the apicoplast. The physiological function of the apicoplast and its integration into parasite metabolism remain poorly understood and at times controversial. We establish that the Toxoplasma apicoplast membrane-localized phosphate translocator (TgAPT) is an essential metabolic link between the endosymbiont and the parasite cytoplasm. TgAPT is required for fatty acid synthesis in the apicoplast, but this may not be its most critical function. Further analyses demonstrate that TgAPT also functions to supply the apicoplast with carbon skeletons for additional pathways and, indirectly, with energy and reduction power. Genetic ablation of the transporter results in rapid death of parasites. The dramatic consequences of loss of its activity suggest that targeting TgAPT could be a viable strategy to identify antiparasitic compounds.

Exogenous nitric oxide triggers Neospora caninum tachyzoite-to-bradyzoite stage conversion in murine epidermal keratinocyte cell cultures.

Neospora caninum, like Toxoplasma gondii, undergoes stage conversion in chronically infected animals, and forms tissue cysts which contain the slowly proliferating bradyzoite stage. These tissue cysts are delineated by a cyst wall, protect the parasite from physiological and immunological reactions on part of the host, and bradyzoites remain viable within an infected host for many years. However, unlike T. gondii, N. caninum bradyzoites have been difficult to obtain using in vitro culture techniques, and current protocols, based on those developed for T. gondii, have been shown to be not very efficient in promoting tachyzoite-to-bradyzoite stage conversion. We report here an alternative in vitro culture method to obtain stage conversion of N. caninum from the proliferative to the cystic stage by using the Nc-Liverpool isolate, murine epidermal keratinocytes as host cells, and continuous treatment of infected cultures with 70 microM sodium nitroprusside for up to 8 days. This treatment significantly reduced parasite proliferation as assessed by Neospora-specific quantitative real-time PCR. The expression of bradyzoite markers was analysed by immunofluorescence following 4 and 8 days of in vitro culture using antibodies directed against bradyzoite antigen 1, the mAbCC2, and the lectin Dolichos biflorus agglutinin. Expression of the tachyzoite-specific immunodominant antigen NcSAG1 and the tachyzoite antigen NcMIC1 was also assessed. Transmission electron microscopy revealed that the majority of parasitophorous vacuoles were in the process of forming a distinct cyst wall through accumulation of granular material at the periphery of the vacuole, and parasites exhibited the typical features of bradyzoites. These findings demonstrate the usefulness of this culture technique as a promising way to study tachyzoite-to-bradyzoite stage conversion in N. caninum in vitro.

Transcriptional profile of Toxoplasma gondii-infected human fibroblasts as revealed by gene-array hybridization.

Abstract. To investigate the host-cell response to infection with the obligate intracellular pathogen Toxoplasma gondii, the transcriptional profiles of infected and uninfected human fibroblasts (HFF) were determined by hybridization to gene arrays representing nearly 600 genes. Transcripts that displayed a greater than five-fold increase in level relative to uninfected controls were also examined by RT-PCR and Northern analysis, resulting in the identification of 13 genes that were strongly up-regulated after infection with T. gondii. Comparisons with the transcriptional profiles of fibroblasts infected with Salmonella typhimurium and Chlamydia trachomatis allowed the identification of genes which are specifically induced in T. gondii-infected cells. While most of the up-regulated genes were induced on infection with all three pathogens, the genes for the transferrin receptor and MacMARCKS were up-regulated in Toxoplasma-infected fibroblasts only. Expression of the transferrin receptor protein was examined by Western analysis and found to be specifically elevated in Toxoplasma-infected fibroblasts. Genes which are specifically induced in T. gondii-infected cells are particularly interesting for further studies, since they might be used to dissect specific interactions of this pathogenic parasite with its host cell.

Type II NADH Dehydrogenase Inhibitor 1-Hydroxy-2-Dodecyl- 4(1H)Quinolone Leads to Collapse of Mitochondrial Inner- Membrane Potential and ATP Depletion in Toxoplasma gondii

ABSTRACT The apicomplexan parasite Toxoplasma gondii expresses type II NADH dehydrogenases (NDH2s) instead of canonical complex I at the inner mitochondrial membrane. These non-proton-pumping enzymes are considered to be promising drug targets due to their absence in mammalian cells. We recently showed by inhibition kinetics that T. gondii NDH2-I is a target of the quinolone-like compound 1-hydroxy-2-dodecyl-4(1H)quinolone (HDQ), which inhibits T. gondii replication in the nanomolar range. In this study, the cationic fluorescent probes Mitotracker and DiOC6(3) (3,3′-dihexyloxacarbocyanine iodine) were used to monitor the influence of HDQ on the mitochondrial inner membrane potential (ΔΨm) in T. gondii. Real-time imaging revealed that nanomolar HDQ concentrations led to a ΔΨm collapse within minutes, which is followed by severe ATP depletions of 30% after 1 h and 70% after 24 h. ΔΨm depolarization was attenuated when substrates for other dehydrogenases that can donate electrons to ubiquinone were added to digitonin-permeabilized cells or when infected cultures were treated with the Fo-ATPase inhibitor oligomycin. A prolonged treatment with sublethal concentrations of HDQ induced differentiation into bradyzoites. This dormant stage is likely to be less dependent on the ΔΨm, since ΔΨm-positive parasites were found at a significantly lower frequency in alkaline-pH-induced bradyzoites than in tachyzoites. Together, our studies reveal that oxidative phosphorylation is essential for maintaining the ATP level in the fast-growing tachyzoite stage and that HDQ interferes with this pathway by inhibiting the electron transport chain at the level of ubiquinone reduction.

Localisation of gluconeogenesis and tricarboxylic acid (TCA)-cycle enzymes and first functional analysis of the TCA cycle in Toxoplasma gondii.

The apicomplexan parasite Toxoplasma gondii displays some unusual localisations of carbohydrate converting enzymes, which is due to the presence of a vestigial, non-photosynthetic plastid, referred to as the apicoplast. It was recently demonstrated that the single pyruvate dehydrogenase complex (PDH) in T. gondii is exclusively localised inside the apicoplast but absent in the mitochondrion. This raises the question about expression, localisation and function of enzymes for the tricarboxylic acid (TCA)-cycle, which normally depends on PDH generated acetyl-CoA. Based on the expression and localisation of epitope-tagged fusion proteins, we show that all analysed TCA cycle enzymes are localised in the mitochondrion, including both isoforms of malate dehydrogenase. The absence of a cytosolic malate dehydrogenase suggests that a typical malate-aspartate shuttle for transfer of reduction equivalents is missing in T. gondii. We also localised various enzymes which catalyse the irreversible steps in gluconeogenesis to a cellular compartment and examined mRNA expression levels for gluconeogenesis and TCA cycle genes between tachyzoites and in vitro bradyzoites. In order to get functional information on the TCA cycle for the parasite energy metabolism, we created a conditional knock-out mutant for the succinyl-CoA synthetase. Disruption of the sixth step in the TCA cycle should leave the biosynthetic parts of the cycle intact, but prevent FADH2 production. The succinyl-CoA synthetase depletion mutant displayed a 30% reduction in growth rate, which could be restored by supplementation with 2 microM succinate in the tissue culture medium. The mitochondrial membrane potential in these parasites was found to be unaltered. The lack of a more severe phenotype suggests that a functional TCA cycle is not essential for T. gondii replication and for maintenance of the mitochondrial membrane potential.

Toxoplasmosis: a persisting challenge

The sixth biennial International Congress on Toxoplasmosis, organized by Uwe Gross (University of Göttingen, Germany), was held on 21-25 May 2001 in Freising, Germany. The first meeting of this kind in 1990 was attended by only 26 investigators and this year there were 115 participants covering various research topics including the immunology, epidemiology, cellular and molecular biology of Toxoplasma gondii.

An 11-bp deletion in the arylsulfatase A gene of a patient with late infantile metachromatic leukodystrophy

SummaryMetachromatic leukodystrophy is a lysosomal storage disorder caused by the deficiency of arylsulfatase A. Examination of the arylsulfatase A gene in a patient suffering from late infantile metachromatic leukodystrophy revealed an 11-bp deletion in exon 8. Although this allele produces normal amounts of ASA mRNA, no arylsulfatase A cross-reacting material could be detected in cultured fibroblasts from the patient. The patient was found to be a compound heterozygote, the other allele is also known to generate no ASA polypeptides. This patient is another example where absence of ASA polypeptides correlates with the severe late infantile form of metachromatic leukodystrophy.

A Hammondia-like parasite from the European fox (Vulpes vulpes) forms biologically viable tissue cysts in cell culture.

Tissue cysts of parasites of the genus Hammondia are rarely described in naturally or experimentally infected intermediate hosts. However, ultrastructural examinations on tissue cyst stages of Hammondia sp. are needed, e.g. to compare these stages with those of Neospora caninum and other related parasites. We describe a cell culture system employed to examine the in vitro development of tissue cysts of a Hammondia sp.-like parasite (isolate FOX 2000/1) which uses the European fox as a definitive host. Cells of a diploid finite cell line from embryonal bovine heart (KH-R; CCLV, RIE 090) were infected by inoculation of sporozoites und cultivated for up to 3 months. Transmission electron microscopic examination of 17 day old cell culture material revealed the presence of cyst walls. Infected cell cultures cultivated for 2 months were used to feed a fox. Six to 13 days post infection the fox shed large numbers (n=1.2 x 10(7)) of Hammondia-sp. like oocysts which could not be distinguished from those used to infect the cell culture as determined by DNA sequencing of the internal transcribed spacer 1 and the D2/D3 domain of the large subunit ribosomal DNA. To find out the proportion of parasitophorous vacuoles that had developed into tissue cysts, the expression of bradyzoite markers was examined by probing infected cell cultures with mouse polyclonal antibodies against Toxoplasma gondii bradyzoite antigen 1 (anti-BAG1) and rat monoclonal antibodies against a cyst wall protein (mAbCC2). Nineteen and 90 days post infection all parasitophorous vacuoles in the cell cultures were positive with anti-BAG1 and mAbCC2. This shows that biologically viable (i.e. infectious) tissue cysts of a fox-derived Hammondia sp. isolate (FOX 2000/1) can be efficiently produced in this cell culture system. Since in vitro cystogenesis of dog-derived Hammondia heydorni has not been observed yet, in vitro cyst formation might be one trait to separate fox-derived Hammondia sp. from H. heydorni on a species level.

Two internal type II NADH dehydrogenases of Toxoplasma gondii are both required for optimal tachyzoite growth

In many apicomplexan parasites the entry of electrons from NADH into the electron transport chain is governed by type II NADH dehydrogenases (NDH2s) instead of a canonical complex I. Toxoplasma gondii expresses two NDH2 isoforms, TgNDH2‐I and TgNDH2‐II with no indication for stage‐specific regulation. We dissected the orientation of both isoforms by using a split GFP assay and a protease protection assay after selective membrane permeabilization. The two approaches revealed that both TgNDH2 isoforms are internal enzymes facing with their active sites to the mitochondrial matrix. Single knockout mutants displayed a decreased replication rate and a reduced mitochondrial membrane potential, which were both more severe in the Tgndh2‐II‐deleted than in the Tgndh2‐I‐deleted mutant. Complementation with a myc‐tagged, ectopic copy of the deleted gene restored the growth rate and the mitochondrial membrane potential. However, an overexpression of the remaining intact isoform could not restore the phenotype, suggesting that the two TgNDH2 isoforms are non‐redundant and possess functional differences. Together, our studies indicate that although TgNDH2‐I and TgNDH2‐II are individually non‐essential, the expression of both internal isoforms is required to maintain the mitochondrial physiology in T. gondii tachyzoites.