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Toxoplasma gondii secretes various virulence effector molecules into host cells to disrupt host interferon-γ (IFN-γ)-dependent immunity. Among these effectors, ROP18 directly phosphorylates and inactivates IFN-inducible GTPases, such as immunity-related GTPases (IRGs) and guanylate-binding proteins (GBPs), leading to the subversion of IFN-inducible GTPase-induced cell-autonomous immunity. The modes of action of ROP18 have been studied extensively; however, little is known about the molecular mechanisms by which ROP18 is produced in the parasite itself. Here, we report the role of T. gondii transcription factor IWS1 in ROP18 mRNA expression in the parasite. Compared with wild-type virulent type I T. gondii, IWS1-deficient parasites showed dramatically increased loading of IRGs and GBPs onto the parasitophorous vacuole membrane (PVM). Moreover, IWS1-deficient parasites displayed decreased virulence in wild-type mice but retained normal virulence in mice lacking the IFN-γ receptor. Furthermore, IWS1-deficient parasites showed severely decreased ROP18 mRNA expression; however, tagged IWS1 did not directly bind with genomic regions of the ROP18 locus. Ectopic expression of ROP18 in IWS1-deficient parasites restored the decreased loading of effectors onto the PVM and in vivo virulence in wild-type mice. Taken together, these data demonstrate that T. gondii IWS1 indirectly regulates ROP18 mRNA expression to determine fitness in IFN-γ-activated host cells and mice. IMPORTANCE The parasite Toxoplasma gondii has a counterdefense system against interferon-γ (IFN-γ)-dependent host immunity which relies on the secretion of parasite effector proteins. ROP18 is one of the effector, which is released into host cells to inactivate IFN-γ-dependent anti-Toxoplasma host proteins. The mechanism by which Toxoplasma ROP18 subverts host immunity has been extensively analyzed, but how Toxoplasma produces this virulence factor remains unclear. Here, we show that Toxoplasma transcription factor IWS1 is important for ROP18 mRNA expression in the parasite. Loss of IWS1 from virulent Toxoplasma leads to dramatically decreased ROP18 mRNA expression, resulting in profoundly decreased virulence due to greater activity of IFN-γ-dependent host immune responses. Thus, Toxoplasma prepares the critical virulence factor ROP18 via an IWS1-dependent system to negate IFN-γ-dependent antiparasitic immunity and thus survive in the host.

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Toxoplasma gondii (T. gondii) causes considerable financial losses in the livestock industry and can present serious threats to pregnant women, as well as immunocompromised patients. Therefore, it is required to design and produce an efficient vaccine for controlling toxoplasmosis. The present study aimed to evaluate the protective immunity induced by RMS protein (ROP18, MIC4, and SAG1) with Freund adjuvant, calcium phosphate nanoparticles (CaPNs), and chitosan nanoparticles (CNs) in BALB/c mice. The RMS protein was expressed in Escherichia coli (E. coli) and purified using a HisTrap HP column. Thereafter, cellular and humoral immunity was assessed by injecting RMS protein on days 0, 21, and 35 into four groups [RMS, RMS-chitosan nanoparticles (RMS-CNs), RMS-calcium phosphate nanoparticles (RMS-CaPNs), and RMS-Freund]. Phosphate buffered saline (PBS), CNs, CaPNs, and Freund served as the four control groups. The results displayed that vaccination with RMS protein and adjuvants significantly elicited the levels of specific IgG antibodies and cytokines against toxoplasmosis. There were high levels of total IgG, IgG2a, and IFN-γ in vaccinated mice, compared to those in the control groups, especially in the RMS-Freund, indicating a Th-1 type response. The vaccinated and control mice were challenged intraperitoneally with 1 × 103 tachyzoites of the T. gondii RH strain four weeks after the last injection, and in RMS-Freund and RMS-CaPNs groups, the highest increase in survival time was observed (15 days). The RMS can significantly increase Th1 and Th2 responses; moreover, multi-epitope vaccines with adjuvants can be a promising strategy for the production of a vaccine against toxoplasmosis.

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Heterologous immunization is garnering attention as a promising strategy to improve vaccine efficacy. Vaccines based on recombinant baculovirus (rBV) and virus-like particle (VLP) are safe for use, but heterologous immunization studies incorporating these two vaccine platforms remain unreported to date. Oral immunization is the simplest, most convenient, and safest means for mass immunization. In the present study, mice were immunized with the Toxoplasma gondii rhoptry protein 18 (ROP18)-expressing rBVs (rBVs-ROP18) and VLPs (VLPs-ROP18) via oral, intranasal, and intramuscular (IM) routes to evaluate the protection elicited against the intracellular parasite T. gondii ME49 strain. Overall, boost immunization with VLPs-ROP18 induced a significant increase in T. gondii-specific antibody response in all three immunization routes. Parasite-specific mucosal and cerebral antibody responses were observed from all immunization groups, but the highest mucosal IgA response was detected from the intestines of orally immunized mice. Antibody-secreting cell (ASC), CD8+ T cell, and germinal center B cell responses were strikingly similar across all three immunization groups. Oral immunization significantly reduced pro-inflammatory cytokine IL-6 in the brains as well as that by IN and IM. Importantly, all of the immunized mice survived against lethal challenge infections where body weight loss was negligible from all three immunizations. These results demonstrated that protection induced against T. gondii by oral rBV-VLP immunization regimen is just as effective as IN or IM immunizations.

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Toxoplasma gondii is an obligate intracellular apicomplexan parasite causing lethal diseases in immunocompromised patients. UBL-UBA shuttle proteins (DDI1, RAD23, and DSK2) are important components of the ubiquitin-proteasome system. By degrading ubiquitinated proteins, UBL-UBA shuttle proteins regulate many cellular processes. However, the specific processes regulated by UBL-UBA shuttle proteins remain elusive. Here, we revealed that the deletion of shuttle proteins results in a selective accumulation of ubiquitinated proteins in the nucleus and aberrant DNA replication. ROP18 was mistargeted and accumulated in the shuttle protein mutant strain, resulting in the recruitment of immunity-related GTPases to the parasitophorous vacuole membrane (PVM). Furthermore, the mistargeting of ROP18 and the recruitment of Irgb6 to the PVM were also observed in the DDI1 mutant strain. DDI1 is a nonclassical UBL-UBA shuttle protein homologous to the HIV-1 protease. Molecular docking showed that DDI1 was a potential target of HIV-1 protease inhibitors. However, these inhibitors blocked the growth of T gondii in vitro but not in vivo. In conclusion, the Toxoplasma UBL-UBA shuttle protein regulates several important cellular processes and the mistargeting of ROP18 may be a representative of the abnormal homeostasis caused by shuttle protein mutation.

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PURPOSE: Antigens expressed by Toxoplasma gondii (T. gondii) during its life cycle trigger various immune responses in the host. Recently, toxoplasma vaccine research focused on T. gondii surface antigen 1 (SAG1) and Rhoptry Protein 18 (ROP18) to establish a safe and efficacious DNA vaccine. METHOD: We constructed two eukaryotic expression plasmids: p3 × FLAG-Myc-CMV™-24-SAG1 and p3 × FLAG-Myc-CMV™-24-ROP18. BALB/c mice were randomly divided into six groups and immunized with these DNA vaccines either separately or in combination. The combination vaccine was administered at either the full dose or at half-strength dose. Control mice were immunized with empty vector or with phosphate-buffered saline. RESULTS: The frequency of CD4+ cells in the spleen was consistent among all groups, whereas that of CD8+ T cells was the highest in the group immunized with the combination vaccine at half-strength dose (p < 0.05). Importantly, the mRNA expression levels of interleukin-12 (IL-12) and interferon-gamma (INF-γ) were closely correlated (r = 0.6, p < 0.0001) and both were upregulated in the group that was immunized with the combination vaccine at half-strength dose (p < 0.0001). The survival time of the mice subjected to a lethal dose of toxoplasma was significantly extended by prior immunization with DNA vaccines expressing either SAG1 or ROP18 or a combination of both (p < 0.05). The group that was immunized with the combination vaccine at half-strength dose demonstrated the best efficacy (p < 0.05). CONCLUSION: These results showed that the combination DNA vaccine provided better immune protection than the single gene vaccines, and that optimizing the dosing of the vaccine can improve the immune response.

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The allelic combination of ROP18/ROP5 genes of Toxoplasma gondii has been shown to be highly predictive of mouse virulence in canonical isolates and strains. The aims of this study were to analyze the alleles present in the ROP18/ROP5 genes from T. gondii isolates obtained in Argentina, to associate the results with the virulence registered in mouse model, and to compare with other isolates and reference strains using a phylogenetic network. Fourteen T. gondii isolates from Argentina were analyzed by nPCR-RFLP for ROP18/ROP5. Phylogenetic network analysis was inferred using the ToxoDB genotypes and the ROPs molecular markers. All isolates and reference strains were categorized as lethal or non-lethal. As results, combinations 2/2, 3/3 and 4/3 for ROP18/ROP5 were detected in 12 isolates, whereas only alleles 1 and 2 of ROP5 were detected in 2 isolates. The majority of the isolates had a mouse virulence matching to that predicted by the ROP18/ROP5 allele combination. The 3 isolates that differed from the expected virulence presented non-clonal genotypes. ROPs incorporation increased the accuracy of the phylogenetic network relations among the T. gondii samples, prevailing the clustering according to regions. Our results indicate a predominance of type 3 allele in both ROP18 and ROP5 markers and an association of allelic profiles 3/3 and 4/3 of non-clonal genotypes from Argentina, both with virulent and avirulent profiles in mice.

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Toxoplasma gondii secretes a number of virulence-related effector proteins, such as the rhoptry protein 18 (ROP18). To further broaden our understanding of the molecular functions of ROP18, we examined the transcriptional response of human embryonic kidney cells (HEK293T) to ROP18 of type I T. gondii RH strain. Using RNA-sequencing, we compared the transcriptome of ROP18-expressing HEK293T cells to control HEK293T cells. Our analysis revealed that ROP18 altered the expression of 750 genes (467 upregulated genes and 283 downregulated genes) in HEK293T cells. Gene ontology (GO) and pathway enrichment analyses showed that differentially expressed genes (DEGs) were significantly enriched in extracellular matrix- and immune-related GO terms and pathways. KEGG pathway enrichment analysis revealed that DEGs were involved in several disease-related pathways, such as nervous system diseases and eye disease. ROP18 significantly increased the alternative splicing pattern "retained intron" and altered the expression of 144 transcription factors (TFs). These results provide new insight into how ROP18 may influence biological processes in the host cells via altering the expression of genes, TFs, and pathways. More in vitro and in vivo studies are required to substantiate these findings.

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BACKGROUND: Toxoplasma gondii is an opportunistic protozoan infecting almost one-third of the world's population. Toxoplasma gondii rhoptry protein 18 (TgROP18) is a key virulence factor determining the parasite's acute virulence and is secreted into host cells during infection. We previously identified the interaction of TgROP18 and host cell immune-related receptor protein IL20RB, and observed the activation of STAT3 in human keratinocytes (HaCaT) cells infected by the rop16 knockout RH strain, though TgROP16 is regarded as being responsible for host STAT3 activation during T. gondii invasion. Therefore, we hypothesize TgROP18 can activate host STAT3 through binding to IL20RB. METHODS: CRISPR-CAS9 technology was used to generate the ROP16 and ROP18 double knockout RH strain, RH-∆rop16∆rop18. SDS-PAGE and western blot were used to detect STAT3 activation in different HaCaT cells with high endogenous IL20RB expression treated with T. gondii tachyzoites infection, recombinant ROP18, or IL-20. FRET and co-immunoprecipitation (Co-IP) was used to detect the protein-protein interaction. RESULTS: We observed that TgROP18 was involved in a synergic activation of the host JAK/STAT3 pathway together with TgROP16 in human HaCaT cells infected with T. gondii or treated with recombinant TgROP18 protein, stimulating host proinflammatory immune responses such as expression of TNF-α. The effect of recombinant ROP18 on STAT3 phosphorylation was presented in a dose-dependent manner. Additionally, TgROP18 was identified to target IL20RB on its extracellular domain. When we treated different cell lines with the recombinant ROP18, STAT3 phosphorylation could only be observed in the cells with endogenous IL20RB expression, such as HaCaT cells. CONCLUSIONS: These findings indicate that TgROP18-IL20RB interaction upon T. gondii invasion was involved in STAT3 activation, which is associated with host cell defense.

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Toxoplasma gondii rhoptry protein TgROP18 is a polymorphic virulence effector that targets immunity-related GTPases (IRGs) in rodents. Given that IRGs are uniquely diversified in rodents and not in other T. gondii intermediate hosts, the role of TgROP18 in manipulating non-rodent cells is unclear. Here we show that in human cells TgROP18I interacts with the interferon-gamma-inducible protein N-myc and STAT interactor (NMI) and that this is a property that is unique to the type I TgROP18 allele. Specifically, when expressed ectopically in mammalian cells only TgROP18I co-immunoprecipitates with NMI in IFN-γ-treated cells, while TgROP18II does not. In parasites expressing TgROP18I or TgROP18II, NMI only co-immunoprecipitates with TgROP18I and this is associated with allele-specific immunolocalization of NMI on the parasitophorous vacuolar membrane (PVM). We also found that TgROP18I reduces NMI association with IFN-γ-activated sequences (GAS) in the IRF1 gene promoter. Finally, we determined that polymorphisms in the C-terminal kinase domain of TgROP18I are required for allele-specific effects on NMI. Together, these data further define new host pathway targeted by TgROP18I and provide the first function driven by allelic differences in the highly polymorphic ROP18 locus.

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The high prevalence of Toxoplasma gondii in the human population in Colombia has been linked to the existence of a high density of urban stray cats, exposing the whole population to a high density of oocysts. The goal of this study was to determine the DNA prevalence of T. gondii by conventional PCR and to phylogenetically analyze ROP18 sequences from positive samples in domestic cat (Felis catus) fecal samples in the city of Armenia, Quindío. Fecal samples from 140 cats were collected from 10 districts around the city. Samples were concentrated using Ritchie's method and analyzed through optical microscopy. Concentrates were used for DNA extraction followed by nested PCR amplification for T. gondii gene B1. PCR for ROP18 was performed on all B1 positive samples; the ROP18 sequences obtained were related to the Archetype I Brazilian and Chinese strains. No oocysts were detected by optical microscopy; however, 17.8% (25/140) B1 and 24% (6/25) ROP18 PCR-positive samples were detected. Phylogenetic analyses showed that isolates clustered into a single group. We assessed whether associations existed between T. gondii positive fecal samples and survey variables such as cat healthcare and socioeconomic characteristics of owners, but no statistically significant associations were found. The presence of T. gondii in cat feces is an important factor contributing to the high prevalence in the human population of this city.

TITLE: Détection d'ADN et génotypes de Toxoplasma gondii dans les fèces de chats domestiques en Colombie. ABSTRACT: La forte prévalence de Toxoplasma gondii dans la population humaine en Colombie a été liée à l'existence d'une forte densité de chats errants urbains, exposant l'ensemble de la population à une forte densité d'oocystes. Le but de ce travail était de déterminer la prévalence de l'ADN de T. gondii par PCR conventionnelle et d'analyser phylogénétiquement les séquences ROP18 d'échantillons positifs dans des échantillons fécaux de chat domestique (Felis catus) dans la ville d'Armenia, Quindío. Des échantillons fécaux de 140 chats ont été collectés dans 10 districts de la ville. Les échantillons ont été concentrés en utilisant la méthode de Ritchie et analysés par microscopie optique. Des concentrés ont été utilisés pour l'extraction d'ADN suivie d'une amplification par PCR nichée pour le gène B1 de T. gondii. La PCR pour ROP18 a été réalisée sur tous les échantillons positifs pour B1 ; les séquences ROP18 obtenues étaient apparentées aux souches Archétype I brésiliennes et chinoises. Aucun oocyste n'a été détecté par microscopie optique mais les échantillons étaient positifs par PCR pour 17,8 % (25/140) pour B1 et 24 % (6/25) pour ROP18. Les analyses phylogénétiques ont montré que les isolats formaient un seul groupe. Nous avons évalué s'il existait des associations entre des échantillons fécaux positifs à T. gondii et des variables d'enquête telles que les soins de santé des chats et les caractéristiques socioéconomiques des propriétaires, mais aucune association statistiquement significative n'a été trouvée. La présence de T. gondii dans les excréments de chats est un facteur important contribuant à la forte prévalence dans la population humaine de cette ville.

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Toxoplasma gondii is a threat for immunocompromized individuals, and no treatment is available for enhancing immunity against infection. Molecular adjuvants may improve the efficacy of DNA vaccine-induced T cell immunity. Here, we report that cocktailed DNA immunization with ROP5 and ROP18 boosted immune responses induced by a single DNA immunization with ROP5 or ROP18, but also that co-administration of molecular adjuvant IL-33 enhanced immune efficacy induced by this cocktailed DNA vaccination. These improved immune responses were characterized by higher Toxoplasma-specific IgG2a titers, Th1 responses associated with the production of IFN-γ, IL-2, IL-12, as well as cell-mediated activity with higher frequencies of CD8+ and CD4+ T cells. More importantly, this enhanced immunity has the ability to confer remarkable protection against a high dose lethal challenge of the T. gondii RH strain and thus against chronic infection with the T. gondii PRU strain. These data show that IL-33 is a promising immunoadjuvant to facilitate humoral as well as cellular immunity in a vaccine setting against T. gondii, and suggest that it should be evaluated in strategies against other apicomplexan parasites.

TITLE: La cytokine IL-33 utilisée comme adjuvant améliore l'immunité protectrice du vaccin à cocktail d'ADN de ROP5 et ROP18 contre l'infection à Toxoplasma gondii chez la souris. ABSTRACT: Toxoplasma gondii est une menace pour les individus immunodéprimés et aucun traitement n'est disponible pour renforcer l'immunité contre l'infection. Les adjuvants moléculaires peuvent améliorer l'efficacité de l'immunité des cellules T induite par un vaccin à ADN. Ici, nous rapportons que l'immunisation par le cocktail d'ADN de ROP5 et ROP18 a stimulé les réponses immunitaires induites par une seule immunisation par l'ADN de ROP5 ou ROP18, mais aussi que la co-administration de l'adjuvant moléculaire IL-33 a amélioré l'efficacité immunitaire induite par cette vaccination par cocktail d'ADN. Ces réponses immunitaires améliorées ont été caractérisées par des titres d'IgG2a spécifiques à Toxoplasma plus élevés, des réponses Th1 associées à la production d'IFN-γ, IL-2, IL-12 ainsi qu'une activité à médiation cellulaire où les fréquences des cellules T CD8+ et CD4+ étaient plus élevées. Plus important encore, cette immunité renforcée a la capacité de conférer une protection remarquable contre une provocation létale par haute dose de la souche RH de T. gondii et donc contre une infection chronique par la souche PRU de T. gondii. Ces données montrent qu'IL-33 est un immunoadjuvant prometteur pour faciliter l'immunité humorale et cellulaire dans un contexte de vaccination contre T. gondii et suggèrent qu'il devrait être évalué dans des stratégies contre d'autres parasites apicomplexes.

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PROPOSE: To report two cases of severe acute multi-systemic failure with bilateral ocular toxoplasmosis in immunocompetent patients from urban settings in Colombia. OBSERVATIONS: We report two immunocompetent male patients aged 44- and 67-years-old who, despite not having visited the Amazonian region in Colombia, had severe bilateral posterior uveitis and extensive-bilateral macular lesions and multiple organ failure that required admission to an intensive care unit. Toxoplasma gondii was positive by PCR assay in vitreous humor samples. Patients were treated with intravitreal clindamycin and dexamethasone in addition to systemic treatment with trimethoprim-sulfamethoxazole. In both patients, infection by atypical strains was confirmed; in one case by serotyping and in another one by genotyping (ROP 18 virulent allele). After 2 and 4 months of treatment respectively, the patients showed improvement of the posterior uveitis and its systemic manifestations. However, there was no significant visual acuity improvement due to bilateral extensive macular involvement. CONCLUSIONS AND IMPORTANCE: Clinicians should be aware that toxoplasmosis originating from South America could be associated with severe acute multisystemic and intraocular bilateral involvement, even in patients with no history of exposure to jungle environments.

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Toxoplasma gondii ROP16 and ROP18 proteins have been identified as important virulence factors for this parasite. Here, we describe the effect of ROP16 and ROP18 proteins on peripheral blood mononuclear cells (PBMCs) from individuals with different clinical status of infection. We evaluated IFN-γ, IL-10, and IL-1ß levels in supernatants from PBMCs cultures infected with tachyzoites of the T. gondii wild-type RH strain or with knock-out mutants of the rop16 and rop18 encoding genes (RHΔrop16 and RHΔrop18). Cytokine secretion was compared between PBMCs obtained from seronegative individuals (n = 10), with those with chronic asymptomatic (n = 8), or ocular infection (n = 12). We also evaluated if polymorphisms in the genes encoding for IFN-γ, IL-10, IL-1ß, Toll-like receptor 9 (TLR9), and purinoreceptor P2RX7 influenced the production of the encoded proteins after ex vivo stimulation. In individuals with chronic asymptomatic infection, only a moderate effect on IL-10 levels was observed when PBMCs were infected with RHΔrop16, whereas a significant difference in the levels of inflammatory cytokines IFN-γ and IL-1ß was observed in seronegative individuals, but this was also dependent on the host's cytokine gene polymorphisms. Infection with ROP16-deficient parasites had a significant effect on IFN-γ production in previously non-infected individuals, suggesting that ROP16 which is considered as a virulence factor plays a role during the primary infection in humans, but not in the secondary immune response.

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Toxoplasma gondii engenders the common parasitic disease toxoplasmosis in almost all warm-blooded animals. Being a critical secretory protein, ROP18 is a major virulence factor of Toxoplasma. There are no reports about ROP18 detection in human serum samples with different clinical manifestations. New aptamers against ROP18 protein were developed through Systematic Evolution of Ligands by Exponential enrichment (SELEX). An Enzyme-Linked Aptamer Assay (ELAA) platform was developed using SELEX-derived aptamers, namely AP001 and AP002. The ELAA was used to evaluate total antigen from T. gondii RH strain (RH Ag) and recombinant protein of ROP18 (rROP18). The results showed that the ELAA presented higher affinity and specificity to RH Ag and rROP18, compared to negative controls. Detection limit of rROP18 protein in serum samples was measured by standard addition method, achieving a lower concentration of 1.56 µg/mL. Moreover, 62 seropositive samples with different clinical manifestations of toxoplasmosis and 20 seronegative samples were tested. A significant association between ELAA test positive for human serum samples and severe congenital toxoplasmosis was found (p = 0.006). Development and testing of aptamers-based assays opens a window for low-cost and rapid tests looking for biomarkers and improves our understanding about the role of ROP18 protein on the pathogenesis of human toxoplasmosis.

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BACKGROUND: Apoptosis plays a critical role in the embryonic development, homeostasis of immune system and host defense against intracellular microbial pathogens. Infection by the obligate intracellular pathogen Toxoplasma gondii can both inhibit and induce host cell apoptosis; however, the parasitic factors involved remain unclear. The T. gondii virulence factor ROP18 (TgROP18) has been reported to regulate host cell apoptosis; nevertheless, results for this regulation have been rarely reported or have provided contradictory findings. Human purinergic receptor 1 (P2X1) is an ATP-gated ion channel that responds to ATP stimulation and functions in cell apoptosis mediation. The precise roles of TgROP18 in T. gondii pathogenesis, and the relationship between TgROP18 and host P2X1 in host cell apoptosis are yet to be revealed. METHODS: Apoptosis rates were determined by flow cytometry (FCM) and TUNEL assay. The interaction between TgROP18 and the host P2X1 was measured by fluorescence resonance energy transfer (FRET) and co-immunoprecipitation (co-IP) assay. Calcium influx and mitochondrial membrane depolarization were determined by FCM after JC-1 staining. The translocation of cytochrome C (Cyt C), Bax and Bcl2 proteins, expression of the apoptotic proteins PARP and caspase activation were detected by western blotting. RESULTS: The apoptosis rates of glial or immune cells (human SF268, mouse RAW264.7 and human THP-1 cells) infected by any T. gondii strain (RH-type I, ME49-type II and VEG-type III) were significantly inhibited compared with their uninfected controls. TgROP18 inhibited ATP-induced apoptosis of SF268 with P2X1 expression, but had no effect on RAW264.7 or THP-1 cells without detectable P2X1 expression. It was further identified that TgROP18 interacted with P2X1, and overexpression of ROP18 in COS7 cells significantly inhibited cell apoptosis mediated by P2X1. Moreover, TgROP18 also inhibited P2X1-mediated Ca2+ influx, translocation of cytochrome C from the mitochondria to the cytosol, and ATP-triggered caspase activation. CONCLUSIONS: Toxoplasma gondii infection inhibits ATP-induced host cell apoptosis, regardless of strain virulence and host cell lines. TgROP18 targets the purinergic receptor P2X1 of the SF268 human neural cells and inhibits ATP-induced apoptosis through the mitochondrial pathway, suggesting a sensor role for the host proapoptotic protein P2X1 in this process.

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BACKGROUND: Rhoptry protein 18 (ROP18) is a key virulence factor of Toxoplasma gondii. The host's immune responses mediated by immune-related GTPases (IRGs) could be blocked by ROP18's kinase activity. ROP18 also interacts with various substrates, such as activating transcription factor 6 beta (ATF6ß) and affects multiple physiological functions within host cells, thereby inducing intense virulence. In this study, competitive inhibitors targeted to ROP18 were subjected to virtual screening based on the principle of structure-based drug design (SBDD). METHODS: The preparation of the ROP18 structure was conducted using the "Structure Prepare" function of Molecular Operating Environment (MOE) software. The ATP-binding pocket was selected as the starting point for virtual screening. Construction of the pharmacophore model used Extended Hückel Theory (EHT) half-quantitative measurement and construction, as well as the characteristics of Type I kinase inhibitors. The pharmacophore model of ROP18 was imported into the Specs database for small molecule similarity screening using EHT pharmacophore measurement. Hit compounds were selected using the functions of London dG and generalized-born volume integral/weighted surface area (GBVI/WSA) scoring. The top 100 hits were analyzed by molecular docking and structure activity relationships (SAR) analysis. RESULTS: The final pharmacophore comprised three typical characteristics: three hydrogen bond acceptors/donors, two ring aromatic features occupying the hydrophobic core, and one cation group feature targeted to the terminus of ATP. A total of 1314 hit compounds analogous to ROP18 pharmacophore were passed through the Specs. After two rounds of docking, 25 out of 100 hits were identified as belonging to two main scaffold types: phthalimide ring structure, thiazole ring and styrene structure. Additionally, the screen also identified 13 inhibitors with distinct scaffold types. The docking models and SAR analysis demonstrated that these hits could engage in multiple hydrogen bonds, salt bridges halogen bonds, and hydrophobic interactions with ROP18, and para-position halo substituents on the benzene ring may enhance their affinity scoring. CONCLUSIONS: A pharmacophore against the ROP18 ATP-binding pocket was successfully constructed, and 25 representative inhibitors from 15 scaffold clusters were screened using the Specs database. Our results provide useful scaffold types for the chemical inhibition of ROP18 or alternative conformations to develop new anti-toxoplasmosis drug leads.

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The severe virulence of Toxoplasma gondii in classical laboratory inbred mouse strains contradicts the hypothesis that house mice (Mus musculus) are the most important intermediate hosts for its transmission and evolution because death of the mouse before parasite transmission equals death of the parasite. However, the classical laboratory inbred mouse strains (Mus musculus domesticus), commonly used to test Toxoplasma strain differences in virulence, do not capture the genetic diversity within Mus musculus. Thus, it is possible that Toxoplasma strains that are severely virulent in laboratory inbred mice are avirulent in some other mouse sub-species. Here, we present insight into the responses of individual mouse strains, representing strains of the genetically divergent Mus musculus musculus, Mus musculus castaneus and Mus musculus domesticus, to infection with individual clonal and atypical Toxoplasma strains. We observed that, unlike M. m. domesticus, M. m. musculus and M. m. castaneus are resistant to the clonal Toxoplasma strains. For M. m. musculus, we show that this is due to a locus on chromosome 11 that includes the genes that encode the interferon gamma (IFNG)-inducible immunity-related GTPases (Irgs) that can kill the parasite by localising and subsequently vesiculating the parasitophorous vacuole membrane. However, despite the localization of known effector Irgs to the Toxoplasma parasitophorous vacuole membrane, we observed that some atypical Toxoplasma strains are virulent in all the mouse strains tested. The virulence of these atypical strains in M. m. musculus could not be attributed to individual rhoptry protein 5 (ROP5) alleles, a secreted parasite pseudokinase that antagonises the canonical effector Irgs and is indispensable for parasite virulence in laboratory inbred mice (M. m. domesticus). We conclude that murine resistance to Toxoplasma is modulated by complex interactions between host and parasite genotypes and may be independent of known effector Irgs on murine chromosome 11.

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Recent studies carried out in Brazil have shown that strains from the same Toxoplasma gondii genotype can infect humans, domestic animals (dogs and cats) and animals slaughtered for human consumption (pigs, sheep, goats, and chickens), suggesting a common infection route. However, little is known about the importance of free-living wild birds within this epidemiological context. The objective of this work was to isolate, genotype, and evaluate the virulence for mice of new isolates of T. gondii obtained from free-living wild birds from the state of Minas Gerais, Southeastern Brazil. From August 2016 to June 2017, T. gondii was isolated from the hearts and brains collected from 6 out of 45 free-living wild birds, namely, a roadside hawk (Rupornis magnirostris), a campo flicker (Colaptes campestris), a southern caracara (Caracara plancus) and a tropical screech-owl (Megascops choliba), all rescued in Belo Horizonte. One isolate was obtained from a toco toucan (Ramphastos toco), rescued in Cristiano Otoni, and another was obtained from southern caracara, rescued in Santa Luzia. Five different genotypes were identified by PCR-RFLP. A unique genotype was shared in two different isolates obtained from a southern caracara and a toco toucan. This genotype has never been previously described in any other host or place. Three isolates were classified as of intermediary virulence and three isolates as avirulent for mice. The combined analysis of alleles ROP18/ROP5 (a serine/threonine kinase, and a polymorphic pseudokinase, respectively) was effective in determining the virulence of five of all the isolates with the exception of that from R. magnirostris. Atypical isolates of T. gondii obtained from free-living wild birds rescued in the state of Minas Gerais share the same genotypes of strains that infect humans, domestic animals, and animals slaughtered for human consumption.

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The neurotropic parasite Toxoplasma gondii is a globally distributed parasitic protozoan among mammalian hosts, including humans. During the course of infection, the CNS is the most commonly damaged organ among invaded tissues. The polymorphic rhoptry protein 18 (ROP18) is a key serine (Ser)/threonine (Thr) kinase that phosphorylates host proteins to modulate acute virulence. However, the basis of neurotropism and the specific substrates through which ROP18 exerts neuropathogenesis remain unknown. Using mass spectrometry, we performed proteomic analysis of proteins that selectively bind to active ROP18 and identified RTN1-C, an endoplasmic reticulum (ER) protein that is preferentially expressed in the CNS. We demonstrated that ROP18 is associated with the N-terminal portion of RTN1-C and specifically phosphorylates RTN1-C at Ser7/134 and Thr4/8/118. ROP18 phosphorylation of RTN1-C triggers ER stress-mediated apoptosis in neural cells. Remarkably, ROP18 phosphorylation of RTN1-C enhances glucose-regulated protein 78 (GRP78) acetylation by attenuating the activity of histone deacetylase (HDAC), and this event is associated with an increase of neural apoptosis. These results clearly demonstrate that both RTN1-C and HDACs are involved in T. gondii ROP18-mediated pathogenesis of encephalitis during Toxoplasma infection.

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RESUMEN

Toxoplasma gondii rhoptry protein ROP18 (TgROP18) is a key virulence factor secreted into the host cell during invasion, where it modulates the host cell response by interacting with its host targets. However, only a few TgROP18 targets have been identified. In this study, we applied a high-throughput protein-protein interaction (PPI) screening in human cells using bimolecular fluorescence complementation (BiFC) to identify the targets of Type I strain ROP18 (ROP18I) and Type II strain ROP18 (ROP18II). From a pool of more than 18,000 human proteins, 492 and 141 proteins were identified as the targets of ROP18I and ROP18II, respectively. Gene ontology, search tool for the retrieval of interacting genes/proteins PPI network, and Ingenuity pathway analyses revealed that the majority of these proteins were associated with immune response and apoptosis. This indicates a key role of TgROP18 in manipulating host's immunity and cell apoptosis, which might contribute to the immune escape and successful parasitism of the parasite. Among the proteins identified, the immunity-related proteins N-myc and STAT interactor, IL20RB, IL21, ubiquitin C, and vimentin and the apoptosis-related protein P2RX1 were further verified as ROP18I targets by sensitized emission-fluorescence resonance energy transfer (SE-FRET) and co-immunoprecipitation. Our study substantially contributes to the current limited knowledge on human targets of TgROP18 and provides a novel tool to investigate the function of parasite effectors in human cells.

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