Cryo-Electron Tomography of Toxoplasma gondii Indicates That the Conoid Fiber May Be Derived from Microtubules.

Toxoplasma gondii (T. gondii) is the causative agent of toxoplasmosis and can infect numerous warm-blooded animals. An improved understanding of the fine structure of this parasite can help elucidate its replication mechanism. Previous studies have resolved the ultrastructure of the cytoskeleton using purified samples, which eliminates their cellular context. Here the application of cryo-electron tomography to visualize T. gondii tachyzoites in their native state is reported. The fine structure and cellular distribution of the cytoskeleton are resolved and analyzed at nanometer resolution. Additionally, the tachyzoite structural characteristics are annotated during its endodyogeny for the first time. By comparing the structural features in mature tachyzoites and their daughter buds, it is proposed that the conoid fiber of the Apicomplexa originates from microtubules. This work represents the detailed molecular anatomy of T. gondii, particularly during the budding replication stage of tachyzoite, and provides a reference for further studies of this fascinating organism.

A comprehensive ultrastructural analysis of the Toxoplasma gondii cytoskeleton.

The invasive nature of Toxoplasma gondii is closely related to the properties of its cytoskeleton, which is constituted by a group of diverse structural and dynamic components that play key roles during the infection. Even if there have been numerous reports about the composition and function of the Toxoplasma cytoskeleton, the ultrastructural organization of some of these components has not yet been fully characterized. This study used a detergent extraction process and several electron microscopy contrast methods that allowed the successful isolation of the cytoskeleton of Toxoplasma tachyzoites. This process allowed for the conservation of the structures known to date and several new structures that had not been characterized at the ultrastructural level. For the first time, characterization was achieved for a group of nanofibers that allow the association between the polar apical ring and the conoid as well as the ultrastructural characterization of the apical cap of the parasite. The ultrastructure and precise location of the peripheral rings were also found, and the annular components of the basal complex were characterized. Finally, through immunoelectron microscopy, the exact spatial location of the subpellicular network inside the internal membrane system that forms the pellicle was found. The findings regarding these new structures contribute to the knowledge concerning the biology of the Toxoplasma gondii cytoskeleton. They also provide new opportunities in the search for therapeutic strategies aimed at these components with the purpose of inhibiting invasion and thus parasitism.

Mitochondria shed their outer membrane in response to infection-induced stress.

The outer mitochondrial membrane (OMM) is essential for cellular homeostasis. Yet little is known of the mechanisms that remodel it during natural stresses. We found that large "SPOTs" (structures positive for OMM) emerge during Toxoplasma gondii infection in mammalian cells. SPOTs mediated the depletion of the OMM proteins mitofusin 1 and 2, which restrict parasite growth. The formation of SPOTs depended on the parasite effector TgMAF1 and the host mitochondrial import receptor TOM70, which is required for optimal parasite proliferation. TOM70 enabled TgMAF1 to interact with the host OMM translocase SAM50. The ablation of SAM50 or the overexpression of an OMM-targeted protein promoted OMM remodeling independently of infection. Thus, Toxoplasma hijacks the formation of SPOTs, a cellular response to OMM stress, to promote its growth.

Computer Aided Image Processing to Facilitate Determination of Congruence in Manual Classification of Mitochondrial Morphologies in Toxoplasma gondii Tissue Cysts.

Toxoplasma gondii is a parasite that chronically infects about a third of the world's population. During chronic infection, the parasite resides within tissue cysts in the form of poorly understood bradyzoites which can number in the thousands. Our prior work showed that these bradyzoites are metabolically active exhibiting heterogeneous replication potential. The morphological plasticity of the mitochondrion potentially informs about parasite metabolic state. We developed an image processing based program to assist manual classification of mitochondrial morphologies by trained operators to collect data and statistics from the manual classification of shapes. We sought to determine whether certain morphologies were readily classifiable and the congruence among manual classifiers, i.e. the degree to which different operators would place the same objects within the same class. Results from three operators classifying mitochondrial morphologies from 5 tissue cyst images showed that among the four classes, one (Blobs) were the easiest to classify. There was remarkable congruence between 2 of the 3 operators in classifying the objects (96%), while the agreement among all 3 operators was somewhat modest (57%). Such information would be valuable for biologists studying these parasites as well as in development of fully automated methods of morphological classification.

Assessment of the Activity of Decoquinate and Its Quinoline-O-Carbamate Derivatives against Toxoplasma gondii In Vitro and in Pregnant Mice Infected with T. gondii Oocysts.

The quinolone decoquinate (DCQ) is widely used in veterinary practice for the treatment of bacterial and parasitic infections, most notably, coccidiosis in poultry and in ruminants. We have investigated the effects of treatment of Toxoplasma gondii in infected human foreskin fibroblasts (HFF) with DCQ. This induced distinct alterations in the parasite mitochondrion within 24 h, which persisted even after long-term (500 nM, 52 days) treatment, although there was no parasiticidal effect. Based on the low half-maximal effective concentration (IC50) of 1.1 nM and the high selectivity index of >5000, the efficacy of oral treatment of pregnant mice experimentally infected with T. gondii oocysts with DCQ at 10 mg/kg/day for 5 days was assessed. However, the treatment had detrimental effects, induced higher neonatal mortality than T. gondii infection alone, and did not prevent vertical transmission. Thus, three quinoline-O-carbamate derivatives of DCQ, anticipated to have better physicochemical properties than DCQ, were assessed in vitro. One such compound, RMB060, displayed an exceedingly low IC50 of 0.07 nM, when applied concomitantly with the infection of host cells and had no impact on HFF viability at 10 µM. As was the case for DCQ, RMB060 treatment resulted in the alteration of the mitochondrial matrix and loss of cristae, but the changes became apparent at just 6 h after the commencement of treatment. After 48 h, RMB060 induced the expression of the bradyzoite antigen BAG1, but TEM did not reveal any other features reminiscent of bradyzoites. The exposure of infected cultures to 300 nM RMB060 for 52 days did not result in the complete killing of all tachyzoites, although mitochondria remained ultrastructurally damaged and there was a slower proliferation rate. The treatment of mice infected with T. gondii oocysts with RMB060 did reduce parasite burden in non-pregnant mice and dams, but vertical transmission to pups could not be prevented.

Toxoplasma LIPIN is essential in channeling host lipid fluxes through membrane biogenesis and lipid storage.

Apicomplexa are obligate intracellular parasites responsible for major human diseases. Their intracellular survival relies on intense lipid synthesis, which fuels membrane biogenesis. Parasite lipids are generated as an essential combination of fatty acids scavenged from the host and de novo synthesized within the parasite apicoplast. The molecular and metabolic mechanisms allowing regulation and channeling of these fatty acid fluxes for intracellular parasite survival are currently unknown. Here, we identify an essential phosphatidic acid phosphatase in Toxoplasma gondii, TgLIPIN, as the central metabolic nexus responsible for controlled lipid synthesis sustaining parasite development. Lipidomics reveal that TgLIPIN controls the synthesis of diacylglycerol and levels of phosphatidic acid that regulates the fine balance of lipids between storage and membrane biogenesis. Using fluxomic approaches, we uncover the first parasite host-scavenged lipidome and show that TgLIPIN prevents parasite death by 'lipotoxicity' through effective channeling of host-scavenged fatty acids to storage triacylglycerols and membrane phospholipids.

Toxoplasma TgATG9 is critical for autophagy and long-term persistence in tissue cysts.

Many of the world's warm-blooded species are chronically infected with Toxoplasma gondii tissue cysts, including an estimated one-third of the global human population. The cellular processes that permit long-term persistence within the cyst are largely unknown for T. gondii and related coccidian parasites that impact human and animal health. Herein, we show that genetic ablation of TgATG9 substantially reduces canonical autophagy and compromises bradyzoite viability. Transmission electron microscopy revealed numerous structural abnormalities occurring in ∆atg9 bradyzoites. Intriguingly, abnormal mitochondrial networks were observed in TgATG9-deficient bradyzoites, some of which contained numerous different cytoplasmic components and organelles. ∆atg9 bradyzoite fitness was drastically compromised in vitro and in mice, with very few brain cysts identified in mice 5 weeks post-infection. Taken together, our data suggests that TgATG9, and by extension autophagy, is critical for cellular homeostasis in bradyzoites and is necessary for long-term persistence within the cyst of this coccidian parasite.

Expansion microscopy provides new insights into the cytoskeleton of malaria parasites including the conservation of a conoid.

Malaria is caused by unicellular Plasmodium parasites. Plasmodium relies on diverse microtubule cytoskeletal structures for its reproduction, multiplication, and dissemination. Due to the small size of this parasite, its cytoskeleton has been primarily observable by electron microscopy (EM). Here, we demonstrate that the nanoscale cytoskeleton organisation is within reach using ultrastructure expansion microscopy (U-ExM). In developing microgametocytes, U-ExM allows monitoring the dynamic assembly of axonemes and concomitant tubulin polyglutamylation in whole cells. In the invasive merozoite and ookinete forms, U-ExM unveils the diversity across Plasmodium stages and species of the subpellicular microtubule arrays that confer cell rigidity. In ookinetes, we additionally identify an apical tubulin ring (ATR) that colocalises with markers of the conoid in related apicomplexan parasites. This tubulin-containing structure was presumed to be lost in Plasmodium despite its crucial role in motility and invasion in other apicomplexans. Here, U-ExM reveals that a divergent and considerably reduced form of the conoid is actually conserved in Plasmodium species.

ATP synthase hexamer assemblies shape cristae of Toxoplasma mitochondria.

Mitochondrial ATP synthase plays a key role in inducing membrane curvature to establish cristae. In Apicomplexa causing diseases such as malaria and toxoplasmosis, an unusual cristae morphology has been observed, but its structural basis is unknown. Here, we report that the apicomplexan ATP synthase assembles into cyclic hexamers, essential to shape their distinct cristae. Cryo-EM was used to determine the structure of the hexamer, which is held together by interactions between parasite-specific subunits in the lumenal region. Overall, we identified 17 apicomplexan-specific subunits, and a minimal and nuclear-encoded subunit-a. The hexamer consists of three dimers with an extensive dimer interface that includes bound cardiolipins and the inhibitor IF1. Cryo-ET and subtomogram averaging revealed that hexamers arrange into ~20-megadalton pentagonal pyramids in the curved apical membrane regions. Knockout of the linker protein ATPTG11 resulted in the loss of pentagonal pyramids with concomitant aberrantly shaped cristae. Together, this demonstrates that the unique macromolecular arrangement is critical for the maintenance of cristae morphology in Apicomplexa.

Biogenesis and discharge of the rhoptries: Key organelles for entry and hijack of host cells by the Apicomplexa.

Rhoptries are specialized secretory organelles found in the Apicomplexa phylum, playing a central role in the establishment of parasitism. The rhoptry content includes membranous as well as proteinaceous materials that are discharged into the host cell in a regulated fashion during parasite entry. A set of rhoptry neck proteins form a RON complex that critically participates in the moving junction formation during invasion. Some of the rhoptry bulb proteins are associated with the membranous materials and contribute to the formation of the parasitophorous vacuole membrane while others are targeted into the host cell including the nucleus to subvert cellular functions. Here, we review the recent studies on Toxoplasma and Plasmodium parasites that shed light on the key steps leading to rhoptry biogenesis, trafficking, and discharge.

Formation of mRNP granules in Toxoplasma gondii during the lytic cycle.

Two poly(A) binding proteins (PABPs) of Toxoplasma gondii, were identified and characterized. They were named TgPABPC and TgPABPN as they were found to localize in the cytoplasm and nucleus respectively. TgPABPC, which colocalizes with mRNA granules, is therefore used as a cellular marker of mRNP granules. We detected that the formation of mRNP granules was independent of polymerized microtubules, and that the granules were distributed stochastically within the cytosol. Formation of mRNP granules was found to occur prior to parasite egress when a Ca2+ ionophore is used to induce egress. It was also found that maturation of mRNP granules could be described as a two-phase process. First, prior to host cell lysis, mRNP granules were formed rapidly within the cytosol. Second, the mRNP granule load was reduced within 10 min post egress. To investigate the link between translational state and mRNP granule formation, treatments with salubrinal, nutrient deprivation, and pH stress were used. While salubrinal induced granule formation in tachyzoites, nutrient starvation and pH stress showed no induction effect on mRNP granule formation. Interestingly, salubrinal treatment in bradyzoites did not induce RNP granule formation, thus suggesting that mRNP granule formation is not a ubiquitous response or directly related to translational repression. Instead, mRNP granule formation is likely a response to the rapid increase in non-translating RNA brought on by sudden changes in translational state.

In vitro effect of a novel protease inhibitor cocktail on Toxoplasma gondii tachyzoites.

Toxoplasmosis is a zoonotic disease and a global food and water-borne infection. The disease is caused by the parasite Toxoplasma gondii, which is a highly successful and remarkable pathogen because of its ability to infect almost any nucleated cell in warm-blooded animals. The present study was done to demonstrate the effect of protease inhibitors cocktail (PIC), which inhibit both cysteine and serine proteases, on in vitro cultured T. gondii tachyzoites on HepG2 cell line. This was achieved by assessing its effect on the invasion of the host cells and the intracellular development of T.gondii tachyzoites through measuring their number and viability after their incubation with PIC. Based on the results of the study, it was evident that the inhibitory action of the PIC was effective when applied to tachyzoites before their cultivation on HepG2 cells. Pre-treatment of T.gondii tachyzoites with PIC resulted in failure of the invasion of most of the tachyzoites and decreased the intracellular multiplication and viability of the tachyzoites that succeeded in the initial invasion process. Ultrastructural studies showed morphological alteration in tachyzoites and disruption in their organelles. This effect was irreversible till the complete lysis of cell monolayer in cultures. It can be concluded that PIC, at in vitro levels, could prevent invasion and intracellular multiplication of Toxoplasma tachyzoites. In addition, it is cost effective compared to individual protease inhibitors. It also had the benefit of combined therapy as it lowered the concentration of each protease inhibitor used in the cocktail. Other in vivo experiments are required to validate the cocktail efficacy against toxoplasmosis. Further studies may be needed to establish the exact mechanism by which the PIC exerts its effect on Toxoplasma tachyzoites behavior and its secretory pathway.

Computational image analysis reveals the structural complexity of Toxoplasma gondii tissue cysts.

Toxoplasma gondii is an obligate intracellular parasite infecting up to one third of the human population. The central event in the pathogenesis of toxoplasmosis is the conversion of tachyzoites into encysted bradyzoites. A novel approach to analyze the structure of in vivo-derived tissue cysts may be the increasingly used computational image analysis. The objective of this study was to quantify the geometrical complexity of T. gondii cysts by morphological, particle, and fractal analysis, as well as to determine if it is impacted by parasite strain, cyst age, and host type. A total of 31 images of T. gondii brain cysts of four type-2 strains (Me49, and local isolates BGD1, BGD14, and BGD26) was analyzed using ImageJ software. The parameters of interest included diameter, circularity, packing density (PD), fractal dimension (FD), and lacunarity. Although cyst diameter varied widely, its negative correlation with PD was observed. Circularity was remarkably close to 1, indicating a perfectly round shape of the cysts. PD and FD did not vary among cysts of different strains, age, and derived from mice of different genetic background. Conversely, lacunarity, which is a measure of heterogeneity, was significantly lower for BGD1 strain vs. all other strains, and higher for Me49 vs. BGD14 and BGD26, but did not differ among Me49 cysts of different age, or those derived from genetically different mice. The results indicate a highly uniform structure and occupancy of the different T. gondii tissue cysts. This study furthers the use of image analysis in describing the structural complexity of T. gondii cyst morphology, and presents the first application of fractal analysis for this purpose. The presented results show that use of a freely available software is a cost-effective approach to advance automated image scoring for T. gondii cysts.

Essential function of the alveolin network in the subpellicular microtubules and conoid assembly in Toxoplasma gondii.

The coccidian subgroup of Apicomplexa possesses an apical complex harboring a conoid, made of unique tubulin polymer fibers. This enigmatic organelle extrudes in extracellular invasive parasites and is associated to the apical polar ring (APR). The APR serves as microtubule-organizing center for the 22 subpellicular microtubules (SPMTs) that are linked to a patchwork of flattened vesicles, via an intricate network composed of alveolins. Here, we capitalize on ultrastructure expansion microscopy (U-ExM) to localize the Toxoplasma gondii Apical Cap protein 9 (AC9) and its partner AC10, identified by BioID, to the alveolin network and intercalated between the SPMTs. Parasites conditionally depleted in AC9 or AC10 replicate normally but are defective in microneme secretion and fail to invade and egress from infected cells. Electron microscopy revealed that the mature parasite mutants are conoidless, while U-ExM highlighted the disorganization of the SPMTs which likely results in the catastrophic loss of APR and conoid.

The zebrafish as a novel model for the in vivo study of Toxoplasma gondii replication and interaction with macrophages.

Toxoplasma gondii is an obligate intracellular parasite capable of invading any nucleated cell. Three main clonal lineages (type I, II, III) exist and murine models have driven the understanding of general and strain-specific immune mechanisms underlying Toxoplasma infection. However, murine models are limited for studying parasite-leukocyte interactions in vivo, and discrepancies exist between cellular immune responses observed in mouse versus human cells. Here, we developed a zebrafish infection model to study the innate immune response to Toxoplasma in vivo By infecting the zebrafish hindbrain ventricle, and using high-resolution microscopy techniques coupled with computer vision-driven automated image analysis, we reveal that Toxoplasma invades brain cells and replicates inside a parasitophorous vacuole to which type I and III parasites recruit host cell mitochondria. We also show that type II and III strains maintain a higher infectious burden than type I strains. To understand how parasites are cleared in vivo, we further analyzed Toxoplasma-macrophage interactions using time-lapse microscopy and three-dimensional correlative light and electron microscopy (3D CLEM). Time-lapse microscopy revealed that macrophages are recruited to the infection site and play a key role in Toxoplasma control. High-resolution 3D CLEM revealed parasitophorous vacuole breakage in brain cells and macrophages in vivo, suggesting that cell-intrinsic mechanisms may be used to destroy the intracellular niche of tachyzoites. Together, our results demonstrate in vivo control of Toxoplasma by macrophages, and highlight the possibility that zebrafish may be further exploited as a novel model system for discoveries within the field of parasite immunity.This article has an associated First Person interview with the first author of the paper.

Division and Adaptation to Host Environment of Apicomplexan Parasites Depend on Apicoplast Lipid Metabolic Plasticity and Host Organelle Remodeling.

Apicomplexan parasites are unicellular eukaryotic pathogens that must obtain and combine lipids from both host cell scavenging and de novo synthesis to maintain parasite propagation and survival within their human host. Major questions on the role and regulation of each lipid source upon fluctuating host nutritional conditions remain unanswered. Characterization of an apicoplast acyltransferase, TgATS2, shows that the apicoplast provides (lyso)phosphatidic acid, required for the recruitment of a critical dynamin (TgDrpC) during parasite cytokinesis. Disruption of TgATS2 also leads parasites to shift metabolic lipid acquisition from de novo synthesis toward host scavenging. We show that both lipid scavenging and de novo synthesis pathways in wild-type parasites exhibit major metabolic and cellular plasticity upon sensing host lipid-deprived environments through concomitant (1) upregulation of de novo fatty acid synthesis capacities in the apicoplast and (2) parasite-driven host remodeling to generate multi-membrane-bound structures from host organelles that are imported toward the parasite.

A doublecortin-domain protein of Toxoplasma and its orthologues bind to and modify the structure and organization of tubulin polymers.

BACKGROUND: TgDCX is a doublecortin-domain protein associated with the conoid fibers, a set of strongly curved non-tubular tubulin-polymers in Toxoplasma. TgDCX deletion impairs conoid structure and parasite invasion. TgDCX contains two tubulin-binding domains: a partial P25α and the DCX/doublecortin domain. Orthologues are found in apicomplexans and their free-living relatives Chromera and Vitrella. RESULTS: We report that isolated TgDCX-containing conoid fibers retain their pronounced curvature, but loss of TgDCX destabilizes the fibers. We crystallized and determined the 3D-structure of the DCX-domain, which is similar to those of human doublecortin and well-conserved among TgDCX orthologues. However, the orthologues vary widely in targeting to the conoid in Toxoplasma and in modulating microtubule organization in Xenopus cells. Several orthologues bind to microtubules in Xenopus cells, but only TgDCX generates short, strongly curved microtubule arcs. EM analysis shows microtubules decorated with TgDCX bundled into rafts, often bordered on one edge by a "C"-shaped incomplete tube. A Chromera orthologue closely mimics TgDCX targeting in Toxoplasma and binds to microtubules in Xenopus cells, but does not generate arcs or "C"-shaped tubes, and fails to rescue the defects of the TgDCX-knockout parasite. CONCLUSIONS: These observations suggest that species-specific features of TgDCX enable it to generate strongly curved tubulin-polymers to support efficient host-cell invasion.

Characterization of the Role of Amylo-Alpha-1,6-Glucosidase Protein in the Infectivity of Toxoplasma gondii.

In this study, we characterized the role of amylo-alpha-1,6-glucosidase (Aa16GL) in the biology and infectivity of Toxoplasma gondii, using Aa16GL-deficient parasites of type I RH and type II Prugniaud (Pru) strains. The subcellular localization of Aa16GL protein was characterized by tagging a 3 × HA to the 3' end of the Aa16GL gene endogenous locus. Immunostaining of the expressed Aa16GL protein revealed that it is located in several small cytoplasmic puncta. Functional characterization of ΔAa16GL mutants using plaque assay, egress assay and intracellular replication assay showed that parasites lacking Aa16GL exhibit a slight reduction in the growth rate, but remained virulent to mice. Although PruΔAa16GL tachyzoites retained the ability to differentiate into bradyzoites in vitro, they exhibited slight reduction in their ability to form cysts in mice. These findings reveal new properties of Aa16GL and suggest that while it does not have a substantial role in mediating T. gondii infectivity, this protein can influence the formation of parasite cysts in mice.

A photoactivatable crosslinking system reveals protein interactions in the Toxoplasma gondii inner membrane complex.

The Toxoplasma gondii inner membrane complex (IMC) is an important organelle involved in parasite motility and replication. The IMC resides beneath the parasite's plasma membrane and is composed of both membrane and cytoskeletal components. Although the protein composition of the IMC is becoming better understood, the protein-protein associations that enable proper functioning of the organelle remain largely unknown. Determining protein interactions in the IMC cytoskeletal network is particularly challenging, as disrupting the cytoskeleton requires conditions that disrupt protein complexes. To circumvent this problem, we demonstrate the application of a photoreactive unnatural amino acid (UAA) crosslinking system to capture protein interactions in the native intracellular environment. In addition to identifying binding partners, the UAA approach maps the binding interface of the bait protein used for crosslinking, providing structural information of the interacting proteins. We apply this technology to the essential IMC protein ILP1 and demonstrate that distinct regions of its C-terminal coiled-coil domain crosslink to the alveolins IMC3 and IMC6, as well as IMC27. We also show that the IMC3 C-terminal domain and the IMC6 N-terminal domain are necessary for binding to ILP1, further mapping interactions between ILP1 and the cytoskeleton. Together, this study develops a new approach to study protein-protein interactions in Toxoplasma and provides the first insight into the architecture of the cytoskeletal network of the apicomplexan IMC.

Biological evaluation of newly synthesized quinoline-based compound PPQ-8 in acute and chronic toxoplasmosis: An experimental study.

Toxoplasma gondii is a widely distributed protozoan parasite, which affects worm-blooded animals including human. The commonest chemotherapeutics used for treatment of symptomatic toxoplasmosis have numerous adverse effects. Thus there is an eminent need to develop new therapeutic agents. Here we described the therapeutic efficacy of 4-(2-chloroquinolin-3-yl)-6-(2,5-dimethoxyphenyl)-2-oxo-1,2-dihydropyridine-3-carbonitrile (PPQ-8); a quinoline-related compound in a mouse model of acute and chronic toxoplasmosis. In acute infection, PPQ-8 decreased the parasite load in liver and spleen with amelioration of the hepatic and splenic pathology. In addition, recovered tachyzoites showed distorted shapes, reduced sizes, irregularities, surface protrusions, erosions and peeling besides apical region distortion when seen by scanning electron microscopy. In chronic toxoplasmosis, PPQ-8 produced degeneration and reduction of the brain cysts without stimulating a damaging inflammatory response within the brain. In both models acute and chronic, PPQ-8 prolonged the survival time of mice. These findings hold promise for the development of a novel anti-toxoplasmosis drug using PPQ-8, but further in vivo studies should be carried out to elucidate PPQ-8 mechanism of action and to report its efficacy in combination with other anti-toxoplasmosis agents.