Glenn A. McConkey

The Neurotropic Parasite Toxoplasma Gondii Increases Dopamine Metabolism

The highly prevalent parasite Toxoplasma gondii manipulates its hosts behavior. In infected rodents, the behavioral changes increase the likelihood that the parasite will be transmitted back to its definitive cat host, an essential step in completion of the parasites life cycle. The mechanism(s) responsible for behavioral changes in the host is unknown but two lines of published evidence suggest that the parasite alters neurotransmitter signal transduction: the disruption of the parasite-induced behavioral changes with medications used to treat psychiatric disease (specifically dopamine antagonists) and identification of a tyrosine hydroxylase encoded in the parasite genome. In this study, infection of mammalian dopaminergic cells with T. gondii enhanced the levels of K+-induced release of dopamine several-fold, with a direct correlation between the number of infected cells and the quantity of dopamine released. Immunostaining brain sections of infected mice with dopamine antibody showed intense staining of encysted parasites. Based on these analyses, T. gondii orchestrates a significant increase in dopamine metabolism in neural cells. Tyrosine hydroxylase, the rate-limiting enzyme for dopamine synthesis, was also found in intracellular tissue cysts in brain tissue with antibodies specific for the parasite-encoded tyrosine hydroxylase. These observations provide a mechanism for parasite-induced behavioral changes. The observed effects on dopamine metabolism could also be relevant in interpreting reports of psychobehavioral changes in toxoplasmosis-infected humans.

A Unique Dual Activity Amino Acid Hydroxylase in Toxoplasma gondii

The genome of the protozoan parasite Toxoplasma gondii was found to contain two genes encoding tyrosine hydroxylase; that produces L-DOPA. The encoded enzymes metabolize phenylalanine as well as tyrosine with substrate preference for tyrosine. Thus the enzymes catabolize phenylalanine to tyrosine and tyrosine to L-DOPA. The catalytic domain descriptive of this class of enzymes is conserved with the parasite enzyme and exhibits similar kinetic properties to metazoan tyrosine hydroxylases, but contains a unique N-terminal extension with a signal sequence motif. One of the genes, TgAaaH1, is constitutively expressed while the other gene, TgAaaH2, is induced during formation of the bradyzoites of the cyst stages of the life cycle. This is the first description of an aromatic amino acid hydroxylase in an apicomplexan parasite. Extensive searching of apicomplexan genome sequences revealed an ortholog in Neospora caninum but not in Eimeria, Cryptosporidium, Theileria, or Plasmodium. Possible role(s) of these bi-functional enzymes during host infection are discussed.

RNA interference (RNAi) inhibits growth of Plasmodium falciparum

RNA interference (RNAi) causes degradation of targeted endogenous RNA in many diverse organisms. Erythrocyte-infecting stages of the malaria parasite Plasmodium falciparum were treated with double-stranded RNA (dsRNA) encoding a segment of the gene encoding dihydroorotate dehydrogenase (DHODH). DHODH is an enzyme in pyrimidine biosynthesis, essential for parasite growth. A decrease in parasite growth (P<0.0005) correlated with a decrease in levels of DHODH mRNA. Control treatments with single-stranded RNA, dsRNA encoding the circumsporozoite protein (a stage-specific protein not expressed in the asexual blood stage) and dsRNA encoding a gene from the related organism Toxoplasma gondii did not inhibit growth. As a test for the RNAi assay, parasites were treated with dsRNA encoding chorismate synthase (CS), an enzyme thought to be involved in folate synthesis, to examine the requirement for this enzyme for parasite growth. Growth decreased (P<0.001) though less markedly than by dsRNA encoding DHODH. These results demonstrate the utility of this assay in assessing requirements for gene products, and their potential as chemotherapeutic targets.

Analysis of short RNAs in the malaria parasite and its red blood cell host

RNA interference (RNAi) is an RNA degradation process that involves short, double‐stranded RNAs (dsRNA) as sequence specificity factors. The natural function of the RNAi machinery is to generate endogenous short double‐stranded RNAs to regulate gene expression. It has been shown that treatment of Plasmodium falciparum, the etiologic agent of malaria, with dsRNA induces degradation of the corresponding microRNA (miRNA), yet typical RNAi‐associated genes have not been identifiable in the parasite genome. To clarify this discrepancy we set out to clone short RNAs from P. falciparum‐infected red blood cells and from purified parasites. We did not find any short RNA that was not a rRNA or tRNA fragment. Indeed, only known human miRNAs were isolated in parasite preparations indicating that very few if any short RNAs exist in P. falciparum. This suggests a different mechanism than classical RNAi in observations of dsRNA‐mediated degradation. Of the human miRNAs identified, the human miRNA mir‐451 accumulates at a very high level in both infected and healthy red blood cells. Interestingly, mir‐451 was not detectable in a series of immortalised cell lines representing progenitor stages of all major blood lineages, suggesting that mir‐451 may play a role in the differentiation of erythroid cells.

Toxoplasma gondii infection and behaviour – location, location, location?

Summary Parasite location has been proposed as an important factor in the behavioural changes observed in rodents infected with the protozoan Toxoplasma gondii. During the chronic stages of infection, encysted parasites are found in the brain but it remains unclear whether the parasite has tropism for specific brain regions. Parasite tissue cysts are found in all brain areas with some, but not all, prior studies reporting higher numbers located in the amygdala and frontal cortex. A stochastic process of parasite location does not, however, seem to explain the distinct and often subtle changes observed in rodent behaviour. One factor that could contribute to the specific changes is increased dopamine production by T. gondii. Recently, it was found that cells encysted with parasites in the brains of experimentally infected rodents have high levels of dopamine and that the parasite encodes a tyrosine hydroxylase, the rate-limiting enzyme in the synthesis of this neurotransmitter. A mechanism is proposed that could explain the behaviour changes due to parasite regulation of dopamine. This could have important implications for T. gondii infections in humans.

Toxoplasma gondii infection, from predation to schizophrenia: can animal behaviour help us understand human behaviour?

Summary We examine the role of the protozoan Toxoplasma gondii as a manipulatory parasite and question what role study of infections in its natural intermediate rodent hosts and other secondary hosts, including humans, may elucidate in terms of the epidemiology, evolution and clinical applications of infection. In particular, we focus on the potential association between T. gondii and schizophrenia. We introduce the novel term ‘T. gondii–rat manipulation–schizophrenia model’ and propose how future behavioural research on this model should be performed from a biological, clinical and ethically appropriate perspective.

Structure-based design, synthesis, and characterization of inhibitors of human and Plasmodium falciparum dihydroorotate dehydrogenases

Pyrimidine biosynthesis is an attractive drug target in a variety of organisms, including humans and the malaria parasite Plasmodium falciparum. Dihydroorotate dehydrogenase, an enzyme catalyzing the only redox reaction of the pyrimidine biosynthesis pathway, is a well-characterized target for chemotherapeutical intervention. In this study, we have applied SPROUT-LeadOpt, a software package for structure-based drug discovery and lead optimization, to improve the binding of the active metabolite of the anti-inflammatory drug leflunomide to the target cavities of the P. falciparum and human dihydroorotate dehydrogenases. Following synthesis of a library of compounds based upon the SPROUT-optimized molecular scaffolds, a series of inhibitors generally showing good inhibitory activity was obtained, in keeping with the SPROUT-LeadOpt predictions. Furthermore, cocrystal structures of five of these SPROUT-designed inhibitors bound in the ubiquinone binding cavity of the human dihydroorotate dehydrogenase are also analyzed.

The transferome of metabolic genes explored: analysis of the horizontal transfer of enzyme encoding genes in unicellular eukaryotes

BackgroundMetabolic networks are responsible for many essential cellular processes, and exhibit a high level of evolutionary conservation from bacteria to eukaryotes. If genes encoding metabolic enzymes are horizontally transferred and are advantageous, they are likely to become fixed. Horizontal gene transfer (HGT) has played a key role in prokaryotic evolution and its importance in eukaryotes is increasingly evident. High levels of endosymbiotic gene transfer (EGT) accompanied the establishment of plastids and mitochondria, and more recent events have allowed further acquisition of bacterial genes. Here, we present the first comprehensive multi-species analysis of E/HGT of genes encoding metabolic enzymes from bacteria to unicellular eukaryotes.ResultsThe phylogenetic trees of 2,257 metabolic enzymes were used to make E/HGT assertions in ten groups of unicellular eukaryotes, revealing the sources and metabolic processes of the transferred genes. Analyses revealed a preference for enzymes encoded by genes gained through horizontal and endosymbiotic transfers to be connected in the metabolic network. Enrichment in particular functional classes was particularly revealing: alongside plastid related processes and carbohydrate metabolism, this highlighted a number of pathways in eukaryotic parasites that are rich in enzymes encoded by transferred genes, and potentially key to pathogenicity. The plant parasites Phytophthora were discovered to have a potential pathway for lipopolysaccharide biosynthesis of E/HGT origin not seen before in eukaryotes outside the Plantae.ConclusionsThe number of enzymes encoded by genes gained through E/HGT has been established, providing insight into functional gain during the evolution of unicellular eukaryotes. In eukaryotic parasites, genes encoding enzymes that have been gained through horizontal transfer may be attractive drug targets if they are part of processes not present in the host, or are significantly diverged from equivalent host enzymes.

metaTIGER: a metabolic evolution resource

Metabolic networks are a subject that has received much attention, but existing web resources do not include extensive phylogenetic information. Phylogenomic approaches (phylogenetics on a genomic scale) have been shown to be effective in the study of evolution and processes like horizontal gene transfer (HGT). To address the lack of phylogenomic information relating to eukaryotic metabolism, metaTIGER (www.bioinformatics.leeds.ac.uk/metatiger) has been created, using genomic information from 121 eukaryotes and 404 prokaryotes and sensitive sequence search techniques to predict the presence of metabolic enzymes. These enzyme sequences were used to create a comprehensive database of 2257 maximum-likelihood phylogenetic trees, some containing over 500 organisms. The trees can be viewed using iTOL, an advanced interactive tree viewer, enabling straightforward interpretation of large trees. Complex high-throughput tree analysis is also available through user-defined queries, allowing the rapid identification of trees of interest, e.g. containing putative HGT events. metaTIGER also provides novel and easy-to-use facilities for viewing and comparing the metabolic networks in different organisms via highlighted pathway images and tables. metaTIGER is demonstrated through evolutionary analysis of Plasmodium, including identification of genes horizontally transferred from chlamydia.

Nucleoside Transport as a Potential Target for Chemotherapy in Malaria

Malaria constitutes an enormous drain on the health and economies of many countries and causes more than a million deaths annually. Moreover, resistance to existing antimalarial drugs is a growing problem, rendering the search for new targets urgent. Protozoan parasites of the genus Plasmodium that cause malaria lack the ability to synthesise the purine ring de novo and so are reliant upon salvage of purines, including hypoxanthine, inosine and adenosine, from the host. The transport systems responsible for uptake of these precursors are therefore promising targets for novel antimalarial drugs. In humans, purine uptake into many cell types is mediated by members of the Equilibrative Nucleoside Transporter (ENT) family, in particular hENT1 and hENT2. Genome sequencing has revealed that P. falciparum and P. vivax, the species responsible for the majority of malaria cases, each also possesses four members of this family, and in P. falciparum transcripts of each are expressed in the erythrocytic stages of the parasite responsible for clinical disease. One of the proteins, PfENT1, is known to be present in the parasite plasma membrane, and the kinetic properties of the heterologously expressed transporter are consistent with its representing the major purine uptake system in the trophozoite. Importantly, its inhibitor specificity and permeant selectivity differ from those of the host. In this review we discuss the possibility of exploiting these differences to develop novel antimalarial drugs that either selectively inhibit purine uptake into the pararasite or are selectively delivered by the transporter to the parasite cytoplasm.