Observing astrocyte polarization in brains from mouse chronically infected with Toxoplasma gondii.

Toxoplasma gondii (T. gondii) is a protozoan parasite that infects approximately one-third of the global human population, often leading to chronic infection. While acute T. gondii infection can cause neural damage in the central nervous system and result in toxoplasmic encephalitis, the consequences of T. gondii chronic infection (TCI) are generally asymptomatic. However, emerging evidence suggests that TCI may be linked to behavioral changes or mental disorders in hosts. Astrocyte polarization, particularly the A1 subtype associated with neuronal apoptosis, has been identified in various neurodegenerative diseases. Nevertheless, the role of astrocyte polarization in TCI still needs to be better understood. This study aimed to establish a mouse model of chronic TCI and examine the transcription and expression levels of glial fibrillary acidic protein (GFAP), C3, C1q, IL-1α, and TNF-α in the brain tissues of the mice. Quantitative real-time PCR (qRT-PCR), enzyme-linked immunosorbent assay, and Western blotting were employed to assess these levels. Additionally, the expression level of the A1 astrocyte-specific marker C3 was evaluated using indirect fluorescent assay (IFA). In mice with TCI, the transcriptional and expression levels of the inflammatory factors C1q, IL-1α, and TNF-α followed an up-down-up pattern, although they remained elevated compared to the control group. These findings suggest a potential association between astrocyte polarization towards the A1 subtype and synchronized changes in these three inflammatory mediators. Furthermore, immunofluorescence assay (IFA) revealed a significant increase in the A1 astrocytes (GFAP+C3+) proportion in TCI mice. This study provides evidence that TCI can induce astrocyte polarization, a biological process that may be influenced by changes in the levels of three inflammatory factors: C1q, IL-1α, and TNF-α. Additionally, the release of neurotoxic substances by A1 astrocytes may be associated with the development of TCI.

Phosphoproteomic analysis reveals changes in A-Raf-related protein phosphorylation in response to Toxoplasma gondii infection in porcine macrophages.

BACKGROUND: Toxoplasma gondii is an obligate intracellular protozoan parasite that causes severe threats to humans and livestock. Macrophages are the cell type preferentially infected by T. gondii in vivo. Protein phosphorylation is an important posttranslational modification involved in diverse cellular functions. A rapidly accelerated fibrosarcoma kinase (A-Raf) is a member of the Raf family of serine/threonine protein kinases that is necessary for MAPK activation. Our previous research found that knockout of A-Raf could reduce T. gondii-induced apoptosis in porcine alveolar macrophages (3D4/21 cells). However, limited information is available on protein phosphorylation variations and the role of A-Raf in macrophages infected with T. gondii. METHODS: We used immobilized metal affinity chromatography (IMAC) in combination with liquid chromatography tandem mass spectrometry (LC-MS/MS) to profile changes in phosphorylation in T. gondii-infected 3D4/21 and 3D4/21-ΔAraf cells. RESULTS: A total of 1647 differentially expressed phosphorylated proteins (DEPPs) with 3876 differentially phosphorylated sites (DPSs) were identified in T. gondii-infected 3D4/21 cells (p3T group) when compared with uninfected 3D4/21 cells (pho3 group), and 959 DEPPs with 1540 DPSs were identified in the p3T group compared with infected 3D4/21-ΔAraf cells (p3KT group). Venn analysis revealed 552 DPSs corresponding to 406 DEPPs with the same phosphorylated sites when comparing p3T/pho3 versus p3T/p3KT, which were identified as DPSs and DEPPs that were directly or indirectly related to A-Raf. CONCLUSIONS: Our results revealed distinct responses of macrophages to T. gondii infection and the potential roles of A-Raf in fighting infection via phosphorylation of crucial proteins.

Toxoplasma gondii modulates the host cell cycle, chromosome segregation, and cytokinesis irrespective of cell type or species origin.

BACKGROUND: Toxoplasma gondii is an apicomplexan intracellular obligate parasite and the etiological agent of toxoplasmosis in humans, domestic animals and wildlife, causing miscarriages and negatively impacting offspring. During its intracellular development, it relies on nutrients from the host cell, controlling several pathways and the cytoskeleton. T. gondii has been proven to control the host cell cycle, mitosis and cytokinesis, depending on the time of infection and the origin of the host cell. However, no data from parallel infection studies have been collected. Given that T. gondii can infect virtually any nucleated cell, including those of humans and animals, understanding the mechanism by which it infects or develops inside the host cell is essential for disease prevention. Therefore, we aimed here to reveal whether this modulation is dependent on a specific cell type or host cell species. METHODS: We used only primary cells from humans and bovines at a maximum of four passages to ensure that all cells were counted with appropriate cell cycle checkpoint control. The cell cycle progression was analysed using fluorescence-activated cell sorting (FACS)-based DNA quantification, and its regulation was followed by the quantification of cyclin B1 (mitosis checkpoint protein). The results demonstrated that all studied host cells except bovine colonic epithelial cells (BCEC) were arrested in the S-phase, and none of them were affected in cyclin B1 expression. Additionally, we used an immunofluorescence assay to track mitosis and cytokinesis in uninfected and T. gondii-infected cells. RESULTS: The results demonstrated that all studied host cell except bovine colonic epithelial cells (BCEC) were arrested in the S-phase, and none of them were affected in cyclin B1 expression. Our findings showed that the analysed cells developed chromosome segregation problems and failed to complete cytokinesis. Also, the number of centrosomes per mitotic pole was increased after infection in all cell types. Therefore, our data suggest that T. gondii modulates the host cell cycle, chromosome segregation and cytokinesis during infection or development regardless of the host cell origin or type.

Apicomplexa micropore: history, function, and formation.

The micropore, a mysterious structure found in apicomplexan species, was recently shown to be essential for nutrient acquisition in Plasmodium falciparum and Toxoplasma gondii. However, the differences between the micropores of these two parasites questions the nature of a general apicomplexan micropore structure and whether the formation process model from Plasmodium can be applied to other apicomplexans. We analyzed the literature on different apicomplexan micropores and found that T. gondii probably harbors a more representative micropore type than the more widely studied ones in Plasmodium. Using recent knowledge of the Kelch 13 (K13) protein interactome and gene depletion phenotypes in the T. gondii micropore, we propose a model of micropore formation, thus enriching our wider understanding of micropore protein function.

The plant-like protein phosphatase PPKL regulates parasite replication and morphology in Toxoplasma gondii.

BACKGROUND: The protozoan parasite Toxoplasma gondii encodes dozens of phosphatases, among which a plant-like phosphatase absent from mammalian genomes named PPKL, which is involved in regulating brassinosteroid signaling in Arabidopsis, was identified in the genome. Among the Apicomplexa parasites, T. gondii is an important and representative pathogen in humans and animals. PPKL was previously identified to modulate the apical integrity and morphology of the ookinetes and parasite motility and transmission in another important parasite, Plasmodium falciparum. However, the exact function of PPKL in the asexual stages of T. gondii remains unknown. METHODS: The plant auxin-inducible degron (AID) system was applied to dissect the phenotypes of PPKL in T. gondii. We first analyzed the phenotypes of the AID parasites at an induction time of 24 h, by staining of different organelles using their corresponding markers. These analyses were further conducted for the parasites grown in auxin for 6 and 12 h using a quantitative approach and for the type II strain ME49 of AID parasites. To further understand the phenotypes, the potential protein interactions were analyzed using a proximity biotin labeling approach. The essential role of PPKL in parasite replication was revealed. RESULTS: PPKL is localized in the apical region and nucleus and partially distributed in the cytoplasm of the parasite. The phenotyping of PPKL showed its essentiality for parasite replication and morphology. Further dissections demonstrate that PPKL is required for the maturation of daughter parasites in the mother cells, resulting in multiple nuclei in a single parasite. The phenotype of the daughter parasites and parasite morphology were observed in another type of T. gondii strain ME49. The substantial defect in parasite replication and morphology could be rescued by genetic complementation, thus supporting its essential function for PPKL in the formation of parasites. The protein interaction analysis showed the potential interaction of PPKL with diverse proteins, thus explaining the importance of PPKL in the parasite. CONCLUSIONS: PPKL plays an important role in the formation of daughter parasites, revealing its subtle involvement in the proper maturation of the daughter parasites during division. Our detailed analysis also demonstrated that depletion of PPKL resulted in elongated tubulin fibers in the parasites. The important roles in the parasites are potentially attributed to the protein interaction mediated by kelch domains on the protein. Taken together, these findings contribute to our understanding of a key phosphatase involved in parasite replication, suggesting the potential of this phosphatase as a pharmaceutic target.

Roles of the tubulin-based cytoskeleton in the Toxoplasma gondii apical complex.

Microtubules (MTs) play a vital role as key components of the eukaryotic cytoskeleton. The phylum Apicomplexa comprises eukaryotic unicellular parasitic organisms defined by the presence of an apical complex which consists of specialized secretory organelles and tubulin-based cytoskeletal elements. One apicomplexan parasite, Toxoplasma gondii, is an omnipresent opportunistic pathogen with significant medical and veterinary implications. To ensure successful infection and widespread dissemination, T. gondii heavily relies on the tubulin structures present in the apical complex. Recent advances in high-resolution imaging, coupled with reverse genetics, have offered deeper insights into the composition, functionality, and dynamics of these tubulin-based structures. The apicomplexan tubulins differ from those of their mammalian hosts, endowing them with unique attributes and susceptibility to specific classes of inhibitory compounds.

Biomechanics of parasite migration within hosts.

The dissemination of protozoan and metazoan parasites through host tissues is hindered by cellular barriers, dense extracellular matrices, and fluid forces in the bloodstream. To overcome these diverse biophysical impediments, parasites implement versatile migratory strategies. Parasite-exerted mechanical forces and upregulation of the host's cellular contractile machinery are the motors for these strategies, and these are comparably better characterized for protozoa than for helminths. Using the examples of the protozoans, Toxoplasma gondii and Plasmodium, and the metazoan, Schistosoma mansoni, we highlight how quantitative tools such as traction force and reflection interference contrast microscopies have improved our understanding of how parasites alter host mechanobiology to promote their migration.

In vitro production of cat-restricted Toxoplasma pre-sexual stages.

Sexual reproduction of Toxoplasma gondii, confined to the felid gut, remains largely uncharted owing to ethical concerns regarding the use of cats as model organisms. Chromatin modifiers dictate the developmental fate of the parasite during its multistage life cycle, but their targeting to stage-specific cistromes is poorly described1,2. Here we found that the transcription factors AP2XII-1 and AP2XI-2 operate during the tachyzoite stage, a hallmark of acute toxoplasmosis, to silence genes necessary for merozoites, a developmental stage critical for subsequent sexual commitment and transmission to the next host, including humans. Their conditional and simultaneous depletion leads to a marked change in the transcriptional program, promoting a full transition from tachyzoites to merozoites. These in vitro-cultured pre-gametes have unique protein markers and undergo typical asexual endopolygenic division cycles. In tachyzoites, AP2XII-1 and AP2XI-2 bind DNA as heterodimers at merozoite promoters and recruit MORC and HDAC3 (ref. 1), thereby limiting chromatin accessibility and transcription. Consequently, the commitment to merogony stems from a profound epigenetic rewiring orchestrated by AP2XII-1 and AP2XI-2. Successful production of merozoites in vitro paves the way for future studies on Toxoplasma sexual development without the need for cat infections and holds promise for the development of therapies to prevent parasite transmission.

Proteomic profile of Toxoplasma gondii stress granules by high-resolution mass spectrometry.

Ribonucleoprotein granules are bio-condensates that form a diverse group of dynamic membrane-less organelles implicated in several cellular functions, including stress response and cellular survival. In Toxoplasma gondii, a type of bio-condensates referred to as stress granules (SGs) are formed prior to the parasites' egress from the host cell and are implicated in the survival and invasion competency of extracellular tachyzoites. We used paraformaldehyde to fix and cross-link SG proteins to allow purification by centrifugation and analysis by mass spectrometry. We profiled protein components of SGs at 10 and 30 min post-egress when parasite's invasion ability is significantly diminished. Thirty-three proteins were identified from 10 min SGs, and additional 43 proteins were identified from 30 min SGs. Notably, common SG components such as proteins with intrinsically disordered domains were not identified. Gene ontology analysis of both 10 and 30 min SGs shows that overall molecular functions of SGs' proteins are ATP-binding, GTP-binding, and GTPase activity. Discernable differences between 10 and 30 min SGs are in the proportions of translation and microtubule-related proteins. Ten-minute SGs have a higher proportion of microtubule-related proteins and a lower proportion of ribosome-related proteins, while a reverse correlation was identified for those of 30 min. It remains to be investigated whether this reverse correlation contributes to the ability of extracellular tachyzoites to reinvade host cells.

Human neutrophil extracellular traps do not impair in vitro Toxoplasma gondii infection.

Introduction: Toxoplasma gondii, responsible for causing toxoplasmosis, is a prevalent food and waterborne pathogen worldwide. It commonly infects warm-blooded animals and affects more than a third of the global human population. Once ingested, the parasite enters the host's small intestine and rapidly disseminates throughout the body via the bloodstream, infiltrating various tissues. Leukocyte-driven responses are vital against T. gondii, with neutrophils playing a dual role: swiftly recruited to infection sites, releasing inflammatory mediators, and serving as a replication hub and Trojan horses, aiding parasite spread. Neutrophils from various hosts release extracellular traps (NETs) against the protozoan. However, gaps persist regarding the mechanisms of NETs production to parasite and their significance in infection control. This study investigates the interplay between human neutrophils and T. gondii, exploring dynamics, key molecules, and signaling pathways involved in NETs production upon protozoan challenge. Methods and Results: Using confocal and electron microscopy, live cell imaging, pharmacological inhibitors, and DNA quantification assays, we find that human neutrophils promptly release both classical and rapid NETs upon pathogen stimulation. The NETs structure exhibits diverse phenotypes over time and is consistently associated with microorganisms. Mechanisms involve neutrophil elastase and peptidylarginine deiminase, along with intracellular calcium signaling and the PI3K pathway. Unexpectedly, human traps do not diminish viability or infectivity, but potentially aid in capturing parasites for subsequent neutrophil phagocytosis and elimination. Discussion: By revealing NETs formation mechanisms and their nuanced impact on T. gondii infection dynamics, our findings contribute to broader insights into host-pathogen relationships.

Global epidemiology and spatial distribution of Toxoplasma gondii in goats: Protocol for a systematic review and Bayesian hierarchical meta-analysis.

BACKGROUND: Toxoplasma gondii, a cosmopolitan protozoan parasite causes toxoplasmosis in humans and many species of domestic and wild animals. T. gondii instigates significant economic losses in sheep and goat farming industry and can lead to abortion, stillbirth, congenital malformations and neonatal losses. The objective of this protocol is to evaluate worldwide seroprevalence of T. gondii exposure in goats using Bayesian hierarchical meta-analysis and geographic information system (GIS). METHODS: A comprehensive literature search will be conducted using search engines, including Web of Science, ScienceDirect, Scopus, PubMed, ProQuest, EMBASE, PROSPERO Register and, Google Scholar without date and language restrictions. The authors search for cross-sectional studies that determine the seroprevalence of T. gondii in goats. Two reviewers will independently screen, selected studies; also, they will extract data, and assess the risk of bias. In case(s) of disagreement, a consensus will be reached with the help of a third author. The Bayesian hierarchical meta-analysis will use to estimate country and worldwide true seroprevalence of T. gondii, which is consist of the sensitivity and specificity of the applied serological assays. The obtained data will be used to identify country-level risk factors associated with T. gondii exposure using GIS in the ArcGIS software. DISCUSSION: The systematic review produced from this protocol will provide the true prevalence rate and spatial distribution T. gondii exposure in goats both regionally and globally using Bayesian hierarchical and GIS analysis. SYSTEMATIC REVIEW REGISTRATION: PROSPERO registration number: CRD42020107928.

Phosphoproteomic Analysis Reveals the Predominating Cellular Processes and the Involved Key Phosphoproteins Essential for the Proliferation of Toxoplasma gondii.

PURPOSE: To explore the essential roles of phosphorylation in mediating the proliferation of T. gondii in its cell lytic life. METHODS: We profiled the phosphoproteome data of T. gondii residing in HFF cells for 2 h and 6 h, representing the early- and late-stages of proliferation (ESP and LSP) within its first generation of division. RESULTS: We identified 70 phosphoproteins, among which 8 phosphoproteins were quantified with the phosphorylation level significantly regulated. While only two of the eight phosphoproteins, GRA7 and TGGT1_242070, were significantly down-regulated at the transcriptional level in the group of LSP vs. ESP. Moreover, GO terms correlated with host membrane component were significantly enriched in the category of cellular component, suggesting phosphoprotein played important roles in acquiring essential substance from host cell via manipulating host membrane. Further GO analysis in the categories of molecular function and biological process and pathway analysis revealed that the cellular processes of glucose and lipid metabolism were regulated by T. gondii phosphoproteins such as PMCAA1, LIPIN, Pyk1 and ALD. Additionally, several phosphoproteins were enriched at the central nodes in the protein-protein interaction network, which may have essential roles in T. gondii proliferation including GAP45, MLC1, fructose-1,6-bisphosphate aldolase, GRAs and so on. CONCLUSION: This study revealed the main cellular processes and key phosphoproteins crucial for the intracellular proliferation of T. gondii, which would provide clues to explore the roles of phosphorylation in regulating the development of tachyzoites and new insight into the mechanism of T. gondii development in vitro.

Cell cycle-regulated ApiAP2s and parasite development: the Toxoplasma paradigm.

The cell division cycle of T. gondii is driven by cyclically expressed ApiAP2 transcription factors (AP2s) that promote gene sets (regulons) associated with specific biological functions. AP2s drive other AP2s, thereby propelling the progressive gene expression waves defining the lytic cycle. AP2s can act as dimers by themselves, in combination with other AP2s (constitutive or cyclical) or in complexes with epigenetic factors. Exit from the cell cycle into either the extracellular state or differentiation into bradyzoites results in major changes in gene expression. Surprisingly, both transitions lead to expression of a shared set of unique AP2s that suggest a shared stress response that, governed by the specific conditions, leads to different outcomes.

Manipulative neuroparasites: uncovering the intricacies of neurological host control.

Manipulative neuroparasites are a fascinating group of organisms that possess the ability to hijack the nervous systems of their hosts, manipulating their behavior in order to enhance their own survival and reproductive success. This review provides an overview of the different strategies employed by manipulative neuroparasites, ranging from viruses to parasitic worms and fungi. By examining specific examples, such as Toxoplasma gondii, Leucochloridium paradoxum, and Ophiocordyceps unilateralis, we highlight the complex mechanisms employed by these parasites to manipulate their hosts' behavior. We explore the mechanisms through which these parasites alter the neural processes and behavior of their hosts, including the modulation of neurotransmitters, hormonal pathways, and neural circuits. This review focuses less on the diseases that neuroparasites induce and more on the process of their neurological manipulation. We also investigate the fundamental mechanisms of host manipulation in the developing field of neuroparasitology, which blends neuroscience and parasitology. Finally, understanding the complex interaction between manipulative neuroparasites and their hosts may help us to better understand the fundamentals of behavior, neurology, and host-parasite relationships.

The effect of chronic experimental toxoplasmosis on some brain neurotransmitters level and behavior changes.

Toxoplasma is capable of causing long-lasting brain cysts in its hosts, which can lead to physiological disturbances in brain neurotransmitters and result in changes in the host's behavior. This study aimed to investigate these changes using an experimental model. Twenty-five female Wistar rats, weighing 220-220 g and six weeks old, were selected for the study. The rats were divided into two control and experimental groups. The experimental group was injected with 5 × 105 tachyzoites of Toxoplasma gondii (virulent RH strain) intra-peritoneally. Four months after the injection, the rats were subjected to behavioral tests, including learning, memory, depression, and locomotor activity tests. The rats were then euthanized, and their brain and serum samples were analyzed for dopamine and serotonin levels. To ensure the presence of cysts in the brain tissue, a PCR test and preparation of pathological slides from the brain tissue were performed. The results showed that the amount of dopamine in the brain of the infected group was significantly higher than that of the control group, while the level of serotonin in brain of the infected group was significantly lower than that of the control group (P < 0.05). However, no significant difference was observed in the amount of these neurotransmitters in the blood of the two groups (P > 0.05). Behavioral changes were evaluated, and it was found that the learning and memory levels of the infected rats were significantly lower than those of the control group (P < 0.05), but no difference was observed in locomotor activity between the two groups (P > 0.05). This experimental infection model indicated that changes in neurotransmitter levels lead to behavior changes. CONCLUSION: The presence of parasite cysts in the brain can affect some of the host's behaviors through changes in neurotransmitter levels. Therefore, there is a possibility that there is a relationship between the presence of Toxoplasma cysts in the brain and neurological disorders. The results of this study suggest that chronic toxoplasmosis may play a role in behavior changes in psychotic diseases.

Factors Influencing Tissue Cyst Yield in a Murine Model of Chronic Toxoplasmosis.

Recent advances into the unique biology of Toxoplasma tissue cysts and the bradyzoites they house necessitate optimization of tissue cyst recovery from infected mouse brains. Here, we present data from 83 tissue cyst purifications of Type II ME49 tissue cysts in CBA/J mice performed over a period of 3 years. The effects of infection with both tissue culture tachyzoites as well as ex vivo tissue cysts were assessed. Significant mortality was restricted to tachyzoite infections with female mice being more susceptible. Infection with tissue cysts was associated with both lower overall symptomology and mortality, exhibiting no sex bias. Cumulatively, host sex did not impact overall tissue cyst yields, although tachyzoite-initiated infections generated significantly higher yields compared to tissue cyst-initiated infections. Notably, serial passage of tissue cysts was accompanied with a decreasing trend for subsequent cyst recovery. The time of tissue cyst harvest, a potential reflection of bradyzoite physiological state, had no significant impact on subsequent cyst yield at the selected time points. In aggregate, these data reveal the considerable heterogeneity associated with tissue cyst yield, making the design of adequately powered experiments critical. This is particularly the case for drug studies where overall tissue cyst burden is currently the primary and often sole metric of efficacy, as the data presented here demonstrate that cyst recovery between preparations of untreated animals can mirror and even exceed the reported effects of drug treatment.

Dimethyl itaconate ameliorates the deficits of goal-directed behavior in Toxoplasma gondii infected mice.

BACKGROUND: The neurotrophic parasite Toxoplasma gondii (T. gondii) has been implicated as a risk factor for neurodegenerative diseases. However, there is only limited information concerning its underlying mechanism and therapeutic strategy. Here, we investigated the effects of T. gondii chronic infection on the goal-directed cognitive behavior in mice. Moreover, we evaluated the preventive and therapeutic effect of dimethyl itaconate on the behavior deficits induced by the parasite. METHODS: The infection model was established by orally infecting the cysts of T. gondii. Dimethyl itaconate was intraperitoneally administered before or after the infection. Y-maze and temporal order memory (TOM) tests were used to evaluate the prefrontal cortex-dependent behavior performance. Golgi staining, transmission electron microscopy, indirect immunofluorescence, western blot, and RNA sequencing were utilized to determine the pathological changes in the prefrontal cortex of mice. RESULTS: We showed that T. gondii infection impaired the prefrontal cortex-dependent goal-directed behavior. The infection significantly downregulated the expression of the genes associated with synaptic transmission, plasticity, and cognitive behavior in the prefrontal cortex of mice. On the contrary, the infection robustly upregulated the expression of activation makers of microglia and astrocytes. In addition, the metabolic phenotype of the prefrontal cortex post infection was characterized by the enhancement of glycolysis and fatty acid oxidation, the blockage of the Krebs cycle, and the disorder of aconitate decarboxylase 1 (ACOD1)-itaconate axis. Notably, the administration of dimethyl itaconate significantly prevented and treated the cognitive impairment induced by T. gondii, which was evidenced by the improvement of behavioral deficits, synaptic ultrastructure lesion and neuroinflammation. CONCLUSION: The present study demonstrates that T. gondii infection induces the deficits of the goal-directed behavior, which is associated with neuroinflammation, the impairment of synaptic ultrastructure, and the metabolic shifts in the prefrontal cortex of mice. Moreover, we report that dimethyl itaconate has the potential to prevent and treat the behavior deficits.

ROP16-mediated activation of STAT6 enhances cyst development of type III Toxoplasma gondii in neurons.

Toxoplasma gondii establishes a long-lived latent infection in the central nervous system (CNS) of its hosts. Reactivation in immunocompromised individuals can lead to life threatening disease. Latent infection is driven by the ability of the parasite to convert from the acute-stage tachyzoite to the latent-stage bradyzoite which resides in long-lived intracellular cysts. While much work has focused on the parasitic factors that drive cyst development, the host factors that influence encystment are not well defined. Here we show that a polymorphic secreted parasite kinase (ROP16), that phosphorylates host cell proteins, mediates efficient encystment of T. gondii in a stress-induced model of encystment and primary neuronal cell cultures (PNCs) in a strain-specific manner. Using short-hairpin RNA (shRNA) knockdowns in human foreskin fibroblasts (HFFs) and PNCs from transgenic mice, we determined that ROP16's cyst enhancing abilities are mediated, in part, by phosphorylation-and therefore activation-of the host cell transcription factor STAT6. To test the role of STAT6 in vivo, we infected wild-type (WT) and STAT6KO mice, finding that, compared to WT mice, STAT6KO mice have a decrease in CNS cyst burden but not overall parasite burden or dissemination to the CNS. Finally, we found a similar ROP16-dependent encystment defect in human pluripotent stem cell-derived neurons. Together, these findings identify a host cell factor (STAT6) that T. gondii manipulates in a strain-specific manner to generate a favorable encystment environment.

Macrophage Migration Inhibitory Factor contributes to drive phenotypic and functional macrophages activation in response to Toxoplasma gondii infection.

Cytokines are small molecules secreted by numerous cells. Macrophage Migration Inhibitory Factor (MIF) is a cytokine initially described due to its function of inhibiting random macrophage migration. Currently, new functions have been described for MIF, such as stimulating inflammatory functions in response to infections by microorganisms including, Toxoplasma gondii. However, the primordial MIF function related to macrophages has been little addressed. The main purpose of the study was to recapitulate MIF function on macrophages in response to T. gondii infection. To achieve this goal, peritoneal macrophages were collected from C57BL/6WT and Mif1-/- mice after recruitment with thioglycolate. Macrophages were cultured, treated with 4-Iodo-6-phenylpyrimidine (4-IPP), and infected or not by T. gondii for 24 h. Following this, the culture supernatant was collected for cytokine, urea and nitrite analysis. In addition, macrophages were evaluated for phagocytic activity and T. gondii proliferation rates. Results demonstrated that T. gondii infection triggered an increase in MIF production in the WT group as well as an increase in the secretion of IL-10, TNF, IFN-γ, IL-6 and IL-17 in the WT and Mif1-/- macrophages. Regarding the comparison between groups, it was detected that Mif1-/- macrophages secreted more IL-10 compared to WT. On the other hand, the WT macrophages produced greater amounts of TNF, IFN-γ, IL-6 and IL-17. Urea production was more pronounced in Mif1-/- macrophages while nitrite production was higher in WT macrophages. T. gondii showed a greater ability to proliferate in Mif1-/- macrophages and these cells also presented enhanced phagocytic activity. In conclusion, T. gondii infection induces macrophage activation inciting cytokine production. In presence of MIF, T. gondii infected macrophages produce pro-inflammatory cytokines compatible with the M1 activation profile. MIF absence caused a dramatic reduction in pro-inflammatory cytokines that are balanced by increased levels of urea and anti-inflammatory cytokines. These macrophages presented increased phagocytic capacity and shared features activation with the M2 profile.

Use of in vitro derived human neuronal models to study host-parasite interactions of Toxoplasma gondii in neurons and neuropathogenesis of chronic toxoplasmosis.

Toxoplasma gondii infects approximately one-third of the world's population resulting in a chronic infection with the parasite located in cysts in neurons in the brain. In most immunocompetent hosts the chronic infection is asymptomatic, but several studies have found correlations between Toxoplasma seropositivity and neuropsychiatric disorders, including Schizophrenia, and some other neurological disorders. Host-parasite interactions of bradyzoites in cysts in neurons is not well understood due in part to the lack of suitable in vitro human neuronal models. The advent of stem cell technologies in which human neurons can be derived in vitro from human induced pluripotent stem cells (hiPSCs) or direct conversion of somatic cells generating induced neurons (iNs), affords the opportunity to develop in vitro human neuronal culture systems to advance the understanding of T. gondii in human neurons. Human neurons derived from hiPSCs or iNs, generate pure human neuron monolayers that express differentiated neuronal characteristics. hiPSCs also generate 3D neuronal models that better recapitulate the cytoarchitecture of the human brain. In this review, an overview of iPSC-derived neurons and iN protocols leading to 2D human neuron cultures and hiPSC-derived 3D cerebral organoids will be given. The potential applications of these 2D and 3D human neuronal models to address questions about host-parasite interactions of T. gondii in neurons and the parasite in the CNS, will be discussed. These human neuronal in vitro models hold the promise to advance the understanding of T. gondii in human neurons and to improve the understanding of neuropathogenesis of chronic toxoplasmosis.