Publisher Correction: Toxoplasma Modulates Signature Pathways of Human Epilepsy, Neurodegeneration & Cancer.

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CSGID Solves Structures and Identifies Phenotypes for Five Enzymes in Toxoplasma gondii.

Toxoplasma gondii, an Apicomplexan parasite, causes significant morbidity and mortality, including severe disease in immunocompromised hosts and devastating congenital disease, with no effective treatment for the bradyzoite stage. To address this, we used the Tropical Disease Research database, crystallography, molecular modeling, and antisense to identify and characterize a range of potential therapeutic targets for toxoplasmosis. Phosphoglycerate mutase II (PGMII), nucleoside diphosphate kinase (NDK), ribulose phosphate 3-epimerase (RPE), ribose-5-phosphate isomerase (RPI), and ornithine aminotransferase (OAT) were structurally characterized. Crystallography revealed insights into the overall structure, protein oligomeric states and molecular details of active sites important for ligand recognition. Literature and molecular modeling suggested potential inhibitors and druggability. The targets were further studied with vivoPMO to interrupt enzyme synthesis, identifying the targets as potentially important to parasitic replication and, therefore, of therapeutic interest. Targeted vivoPMO resulted in statistically significant perturbation of parasite replication without concomitant host cell toxicity, consistent with a previous CRISPR/Cas9 screen showing PGM, RPE, and RPI contribute to parasite fitness. PGM, RPE, and RPI have the greatest promise for affecting replication in tachyzoites. These targets are shared between other medically important parasites and may have wider therapeutic potential.

Toxoplasma Modulates Signature Pathways of Human Epilepsy, Neurodegeneration & Cancer.

One third of humans are infected lifelong with the brain-dwelling, protozoan parasite, Toxoplasma gondii. Approximately fifteen million of these have congenital toxoplasmosis. Although neurobehavioral disease is associated with seropositivity, causality is unproven. To better understand what this parasite does to human brains, we performed a comprehensive systems analysis of the infected brain: We identified susceptibility genes for congenital toxoplasmosis in our cohort of infected humans and found these genes are expressed in human brain. Transcriptomic and quantitative proteomic analyses of infected human, primary, neuronal stem and monocytic cells revealed effects on neurodevelopment and plasticity in neural, immune, and endocrine networks. These findings were supported by identification of protein and miRNA biomarkers in sera of ill children reflecting brain damage and T. gondii infection. These data were deconvoluted using three systems biology approaches: "Orbital-deconvolution" elucidated upstream, regulatory pathways interconnecting human susceptibility genes, biomarkers, proteomes, and transcriptomes. "Cluster-deconvolution" revealed visual protein-protein interaction clusters involved in processes affecting brain functions and circuitry, including lipid metabolism, leukocyte migration and olfaction. Finally, "disease-deconvolution" identified associations between the parasite-brain interactions and epilepsy, movement disorders, Alzheimer's disease, and cancer. This "reconstruction-deconvolution" logic provides templates of progenitor cells' potentiating effects, and components affecting human brain parasitism and diseases.

Membrane skeletal association and post-translational allosteric regulation of Toxoplasma gondii GAPDH1.

When Toxoplasma gondii egresses from the host cell, glyceraldehyde-3-phosphate dehydrogenase 1 (GAPDH1), which is primary a glycolysis enzyme but actually a quintessential multifunctional protein, translocates to the unique cortical membrane skeleton. Here, we report the 2.25 Å resolution crystal structure of the GAPDH1 holoenzyme in a quaternary complex providing the basis for the molecular dissection of GAPDH1 structure-function relationships Knockdown of GAPDH1 expression and catalytic site disruption validate the essentiality of GAPDH1 in intracellular replication but we confirmed that glycolysis is not strictly essential. We identify, for the first time, S-loop phosphorylation as a novel, critical regulator of enzymatic activity that is consistent with the notion that the S-loop is critical for cofactor binding, allosteric activation and oligomerization. We show that neither enzymatic activity nor phosphorylation state correlate with the ability to translocate to the cortex. However, we demonstrate that association of GAPDH1 with the cortex is mediated by the N-terminus, likely palmitoylation. Overall, glycolysis and cortical translocation are functionally decoupled by post-translational modifications.

The structure of bradyzoite-specific enolase from Toxoplasma gondii reveals insights into its dual cytoplasmic and nuclear functions.

In addition to catalyzing a central step in glycolysis, enolase assumes a remarkably diverse set of secondary functions in different organisms, including transcription regulation as documented for the oncogene c-Myc promoter-binding protein 1. The apicomplexan parasite Toxoplasma gondii differentially expresses two nuclear-localized, plant-like enolases: enolase 1 (TgENO1) in the latent bradyzoite cyst stage and enolase 2 (TgENO2) in the rapidly replicative tachyzoite stage. A 2.75 Šresolution crystal structure of bradyzoite enolase 1, the second structure to be reported of a bradyzoite-specific protein in Toxoplasma, captures an open conformational state and reveals that distinctive plant-like insertions are located on surface loops. The enolase 1 structure reveals that a unique residue, Glu164, in catalytic loop 2 may account for the lower activity of this cyst-stage isozyme. Recombinant TgENO1 specifically binds to a TTTTCT DNA motif present in the cyst matrix antigen 1 (TgMAG1) gene promoter as demonstrated by gel retardation. Furthermore, direct physical interactions of both nuclear TgENO1 and TgENO2 with the TgMAG1 gene promoter are demonstrated in vivo using chromatin immunoprecipitation (ChIP) assays. Structural and biochemical studies reveal that T. gondii enolase functions are multifaceted, including the coordination of gene regulation in parasitic stage development. Enolase 1 provides a potential lead in the design of drugs against Toxoplasma brain cysts.

Structural and functional divergence of the aldolase fold in Toxoplasma gondii.

Parasites of the phylum Apicomplexa are highly successful pathogens of humans and animals worldwide. As obligate intracellular parasites, they have significant energy requirements for invasion and gliding motility that are supplied by various metabolic pathways. Aldolases have emerged as key enzymes involved in these pathways, and all apicomplexans express one or both of fructose 1,6-bisphosphate (F16BP) aldolase and 2-deoxyribose 5-phosphate (dR5P) aldolase (DERA). Intriguingly, Toxoplasma gondii, a highly successful apicomplexan parasite, expresses F16BP aldolase (TgALD1), d5RP aldolase (TgDERA), and a divergent dR5P aldolase-like protein (TgDPA) exclusively in the latent bradyzoite stage. While the importance of TgALD1 in glycolysis is well established and TgDERA is also likely to be involved in parasite metabolism, the detailed function of TgDPA remains elusive. To gain mechanistic insight into the function of different T. gondii aldolases, we first determined the crystal structures of TgALD1 and TgDPA. Structural analysis revealed that both aldolases adopt a TIM barrel fold accessorized with divergent secondary structure elements. Structural comparison of TgALD1 and TgDPA with members of their respective enzyme families revealed that, while the active-site residues are conserved in TgALD1, key catalytic residues are absent in TgDPA. Consistent with this observation, biochemical assays showed that, while TgALD1 was active on F16BP, TgDPA was inactive on dR5P. Intriguingly, both aldolases are competent to bind polymerized actin in vitro. Altogether, structural and biochemical analyses of T. gondii aldolase and aldolase-like proteins reveal diverse functionalization of the classic TIM barrel aldolase fold.

Are rhoptries in Apicomplexan parasites secretory granules or secretory lysosomal granules?

The club-shaped rhoptries in Apicomplexan parasites are one of the most unusual secretory organelles among the eukaryotes, containing unusual lipid and protein cargo that is specialized for intracellular parasitism. Rhoptries have traditionally been viewed strictly as regulated secretory granules. We discuss in this article recent data on the cargo, function and biogenesis of rhoptries in two parasitic model systems, Toxoplasma and Plasmodium. Current findings suggest that rhoptries receive products from both biosynthetic and endocytic pathways and, therefore, they are most analogous to secretory lysosomal granules found in mammalian cells.

Pleiotropic effect due to targeted depletion of secretory rhoptry protein ROP2 in Toxoplasma gondii.

Long after their discovery, the function and biogenesis of rhoptries remain enigmatic. In Apicomplexan parasites, these organelles discharge and their contents are exocytosed at the time of host cell invasion, and are thus proposed to play an essential role in establishing the parasitophorous vacuole. In Toxoplasma gondii, ROP2 is suspected to serve as the molecular link between host cell mitochondria and parasitophorous vacuole membrane. In this study we addressed the function of ROP2. Targeted depletion of ROP2 using a ribozyme-modified antisense RNA strategy resulted in multiple effects on parasite morphology because of a disruption in the formation of mature rhoptries, and an arrest in cytokinesis. The association of host cell mitochondria with the parasitophorous vacuole membrane was abolished and the ROP2-deficient parasites had a reduced uptake of sterol from the host cell. Furthermore, these parasites invaded human fibroblasts poorly and had markedly attenuated virulence in mice. We conclude that rhoptry discharge, and in particular release of ROP2, are essential for parasite invasion, replication and host cell-parasite interaction.

AP-1 in Toxoplasma gondii mediates biogenesis of the rhoptry secretory organelle from a post-Golgi compartment.

We have previously demonstrated that Toxoplasma gondii has a tyrosine-based sorting system, which mediates protein targeting to the lysosome-like rhoptry secretory organelle. We now show that rhoptry protein targeting is also dependent on a dileucine motif and occurs from a post-Golgi endocytic organelle to mature rhoptries in an adaptin-dependent fashion. The T. gondii AP-1 adaptin complex is implicated in this transport because the micro1 chain of T. gondii AP-1 (a) was localized to multivesicular endosomes and the limiting and luminal membranes of the rhoptries; (b) bound to endocytic tyrosine motifs in rhoptry proteins, but not in proteins from dense granule secretory organelles; (c) when mutated in predicted tyrosine-binding motifs, led to accumulation of the rhoptry protein ROP2 in a post-Golgi multivesicular compartment; and (d) when depleted via antisense mRNA, resulted in accumulation of multivesicular endosomes and immature rhoptries. These are the first results to implicate AP-1 in transport from a post-Golgi compartment to a mature secretory organelle and substantially expand the role for AP-1 in anterograde protein transport.

Golgi biogenesis in Toxoplasma gondii.

Two models have been put forward to explain the growth of new Golgi during the cell cycle. The first suggests that a new Golgi grows out of the endoplasmic reticulum by de novo synthesis. The second suggests that a pre-existing Golgi is needed for the growth of a new one, that is, the Golgi is an autonomously replicating organelle. To resolve this issue, we have exploited the simplicity of the apicomplexan parasite Toxoplasma gondii, which has only a single Golgi stack. Here we show, by using video fluorescence microscopy and three-dimensional reconstructions of serial thin sections, that the Golgi grows by a process of lateral extension followed by medial fission. Further fission leads to the inheritance by each daughter of a pair of Golgi structures, which then coalesce to re-form a single Golgi. Our results indicate that new Golgi grow by autonomous duplication and raise the possibility that the Golgi is a paired structure that is analogous to centrioles.

Toxoplasma gondii Rab5 enhances cholesterol acquisition from host cells.

The role of endocytosis in nutrient uptake by Toxoplasma gondii is unknown. To explore this issue, we characterized an endosomal compartment by identifying a T. gondii Rab5 homologue, a molecular marker for early endosomes in eukaryotic cells. The deduced amino acid sequence of the T. gondii Rab5 gene encodes a protein of 240 amino acids, which we termed TgRab51. TgRab51 was epitope-tagged at the N-terminus, expressed in the parasite, and localized by immunofluorescence and immunoelectron microscopy to tubulovesicular structures anterior to the parasite nucleus and adjacent to, but distinct from the Golgi. By immunofluorescence analysis, TgRab51wt-HA staining partially overlapped with Golgi/TGN markers, but not with the T. gondii secretory organelles. A dominant positive mutant, TgRab51Q103L-HA, enhanced uptake of exogenous cholesterol analogues in intracellular parasites, augmented formation of lipid droplets and accelerated parasite growth. Brefeldin A disrupted the TgRab51 compartment, and altered the distribution of fluorescent exogenous cholesterol in cells expressing TgRab51Q103L-HA. These results suggest that TgRab51 facilitates sterol uptake, possibly through a Golgi-dependent pathway.