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The present study aimed to evaluate a DNA vector plasmid encoding a partial rop18 gene from Toxoplasma gondii in domestic cats as a potential vaccine candidate. Four domestic cats (Felis catus) were used, of which two animals received 25 μg of pcDNA 3.1 + rop18, and two received 25 μg of pcDNA 3.1. All animals received intramuscular immunizations with four doses every three weeks along with 1.5 % levamisole. Thirty days after the last immunization, the animals were infected with 300 tissue cysts from ToxoDB #182 strain, a non-archetypal genotype isolated from a wild cat. Fecal examinations were performed for oocyst shedding. Enzyme-linked immunosorbent assay and western blotting analyses with recombinant ROP18 were performed to assess the humoral immune response. Animals that received plasmid containing the partial T. gondii rop18 gene produced specific IgG antibodies and shed 53.3 % fewer oocysts than controls. The two groups of animals showed no statistically significant differences (p > 0.05) in oocyst shedding; however, they showed significant differences in the detection of anti-Toxoplasma antibodies (p < 0.05). In conclusion, the T. gondii rop18 gene is a potential vaccine candidate against oocyst shedding in cats.

engenders the common parasitic disease toxoplasmosis in almost all warm-blooded animals. Being a critical secretory protein, ROP18 is a major virulence factor of . 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 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 ( = 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.

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.

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. 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.

Rhoptry protein 18 (ROP18) is a key virulence factor secreted into host cells during the invasion of Toxoplasma gondii (T. gondii) and plays an important role in the pathogenesis of infection. Due to its potential as a vaccine candidate, this study aimed to characterize several properties of the T. gondii ROP18 (TgROP18) protein to support its inclusion in vaccine formulations. Using a range of bioinformatics tools, we investigated its T-cell and B-cell epitopes, physicochemical properties, subcellular localization, transmembrane domains, and tertiary and secondary structures. Our analysis revealed 48 post-translational modification sites in TgROP18. The secondary structure was composed of 4.87% beta-turns, 38.45% random coils, 42.42% alpha helices, and 14.26% extended strands. Several potential T- and B-cell epitopes were identified on ROP18. The Ramachandran plot of both crude and refined models showed that 85.8% and 95.3% of the amino acid residues, respectively, fell within favored regions, indicating energetically stable conformations. Allergenicity and antigenicity assessments indicated that TgROP18 is a nonallergenic, immunogenic protein. Predictions using the C-ImmSim server suggest that TgROP18 can stimulate humoral and cell-mediated immune responses, based on antibody titers and cytokine profiles following antigen administration. These findings provide baseline data for future investigations focused on the potential of TgROP18 in developing therapeutic strategies against toxoplasmosis.

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-1β levels in supernatants from PBMCs cultures infected with tachyzoites of the wild-type RH strain or with knock-out mutants of the and encoding genes (RHΔ and RHΔ ). Cytokine secretion was compared between PBMCs obtained from seronegative individuals ( = 10), with those with chronic asymptomatic ( = 8), or ocular infection ( = 12). We also evaluated if polymorphisms in the genes encoding for -γ, β, Toll-like receptor 9 ( ), and purinoreceptor influenced the production of the encoded proteins after stimulation. In individuals with chronic asymptomatic infection, only a moderate effect on IL-10 levels was observed when PBMCs were infected with RHΔ , whereas a significant difference in the levels of inflammatory cytokines IFN-γ and IL-1β 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.

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.

Background Decidual macrophages (dMφ) are pivotal in maintaining maternal–fetal immune tolerance during normal pregnancy by expressing a range of immune-suppressive molecules, including CD73. It has been demonstrated that Toxoplasma gondii ( T . gondii ) infection during pregnancy can impair dMφ function, potentially leading to adverse pregnancy outcomes, through downregulation of these inhibitory molecules. T . gondii rhoptry protein 18 ( Tg ROP18), a key virulence factor of T . gondii , is associated with the incapacitation of the host’s innate and adaptive immune responses to protect the parasite from elimination. However, the role of Tg ROP18 in modulating CD73 expression on dMφ and the underlying mechanisms remain to be elucidated. Methods Wild-type (WT) and CD73-deficient (CD73 −/− ) pregnant mice were subjected to intraperitoneal injection of T . gondii RH or RH-Δ rop18 on gestational day (Gd) 8, and subsequently euthanized on Gd 14. Pregnancy outcomes were then evaluated, and the expression levels of CD73, arginase 1 (Arg-1), and interleukin 10 (IL-10) were quantified by flow cytometry. Mononuclear cells isolated from the human aborted decidual tissues were also infected with T . gondii RH or RH-Δ rop18 for the analysis of CD73 expression with flow cytometry. Additionally, infected human dMφ were used to assess the expression levels of CD73, Arg-1, IL-10, and their associated signaling molecules by western blot analysis. Furthermore, chromatin immunoprecipitation (ChIP) assays were performed to validate the involved signaling pathways. Results Compared with the T . gondii RH-infected group, milder adverse pregnancy outcomes and attenuated expression levels of CD73 on dMφ were observed in T . gondii RH-Δ rop18 -infected pregnant mice and human decidual tissues. Lysine-specific histone demethylase1 (LSD1) and snail family transcriptional repressor 1 (SNAIL1) were found to be involved in the downregulation of CD73 expression on dMφ following T . gondii infection. Subsequently, reduced expression of CD73 contribute to the downregulation of Arg-1 and IL-10 expression through adenosine A2a receptor (A2AR) / protein kinase A (PKA) / phosphorylated cAMP-response element binding protein (p-CREB) / CCAAT enhancer binding protein B (C/EBPβ) pathway. Conclusions Tg ROP18 can significantly reduce CD73 expression on dMφ through LSD1/SNAIL1 pathway, subsequently leading to the decreased expression levels of Arg-1 and IL-10 via adenosine/A2AR/PKA/p-CREB/C/EBPβ pathway, which ultimately contributes to maternal–fetal tolerance dysfunction of dMφ. Graphical Abstract

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.

Toxoplasmosis is one of the most common zoonotic protozoal diseases. Recent advances in biotechnology have produced recombinant protein, which are immunogenic, and progress in nano-pharmaceutics has generated encapsulated protein in nanospheres, which are suitable for vaccine delivery. DNA was extracted from Toxoplasma gondii oocysts and was confirmed through nested PCR and sequencing. The 1665 bp of ROP18 was cloned into the easy vector system: pGEM-T by the T-A cloning method. DH5α bacteria were transfected with pGEM-ROP18. ROP18 was subcloned from pGEM-ROP18 into pET28-ROP18. BL21 bacteria were transfected with pET28-ROP18. Thus, rROP18 protein was expressed in BL21 bacteria by induction at different concentrations of isopropyl β-D-1-thiogalactopyranoside. Protein expression was confirmed through SDS-PAGE and Western blotting. The immunoblot of rROP18 was recognized by anti-HIS antibodies and sera from infected mice at 67 kDa. Recombinant ROP18 protein was encapsulated in nanoparticles with PLGA and was characterized through scanning electron microscopy. Intraperitoneal immunizations with rROP18 protein and intranasal immunization of nanospheres were carried out in mice, and the immune response was detected by ELISA. Results showed that rROP18 in nanospheres administered intra-nasally elicited elevated responses of specific IgA and IgG2a as compared to groups inoculated intra-nasally with rROP18 alone, or injected subcutaneously with rROP18 in montanide adjuvant. It was concluded that nanospheres of ROP18 would be a non-invasive approach to develop vaccination against T. gondii. Further experiments are needed to determine the cellular response to these nanospheres in a mouse model for chronic toxoplasmosis.