Population-based cohort study of Toxoplasma gondii P22 antibody positivity correlation with anxiety.

BACKGROUND: Accumulating evidence suggests that latent infection with Toxoplasma gondii (T. gondii) is associated with a variety of neuropsychiatric and behavioral conditions. This research aims to explore the potential correlation between T. gondii antibody positivity and neuropsychiatric disorders through a comprehensive prospective cohort study. METHODS: The cohort study utilized the UK Biobank database to recruit 8814 individuals with no prior diagnosis of neuropsychiatric disorders. Cox proportional hazards models were employed to investigate the associations between T. gondii P22 antibody seropositivity (P22+) and the development of various types of neuropsychiatric disorders. RESULTS: Of the population, 14.65 % tested positive for T. gondii P22 antibody. The presence of T. gondii P22 antibody showed a slight inverse association with epilepsy (HR: 0.28; 95 % CI: 0.10-0.77), while it was positively associated with an increased risk of developing anxiety disorders (HR: 1.38; 95 % CI: 1.04-1.83). LIMITATIONS: The study sample consisted mostly of white British individuals aged 40 to 69 years old. Although we adjusted for potential confounders, there may be other unmeasured and residual confounding factors that could have influenced our reported associations. CONCLUSIONS: The findings suggested an increased risk of anxiety and potential evidence of epilepsy associated with T. gondii P22+. However, our analysis did not reveal an increased risk of several other neuropsychiatric conditions including Alzheimer's disease, dementia, substance abuse disorders, depression, and neurodegenerative disorders, associated with P22 antibody seropositivity.

Toxoplasma gondii infection positively associated with schizophrenia: Evidences from UK Biobank cohort and case-controlled studies.

Toxoplasma gondii (T. gondii) is an opportunistic pathogen affecting about 1/3 of world population. While often asymptomatic in immunocompetent individuals, it can lead to severe toxoplasmosis in immunocompromised patients. Recent research has unveiled a potential link between T. gondii infection and neuropsychiatric diseases. We implemented both a cohort study and a case control study to further identify this association. In the cohort study, we analyzed data from the UK Biobank database, which included 8814 individuals tested for T. gondii SAG1 antibodies and free of neuropsychiatric disorders at baseline. Among them, 22.52% (n = 1985) tested positive for SAG1 antibody. Over an average follow-up period of 12.26 years, Cox proportional hazards models and logistic regression analysis revealed a significant association between the SAG1 seropositivity at baseline and the incidence of schizophrenia (HR: 5.89; 95% CI: 1.69-20.53). In our case-control study, 239 patients diagnosed with schizophrenia and 455 healthy individuals were involved. Using the modified agglutination test (MAT) to detect T. gondii antibodies, logistic regression analysis showed a higher prevalence of T. gondii infection among schizophrenia patients (10.04%) compared to healthy controls (3.74%). T. gondii infection emerged as a significant risk factor for schizophrenia (OR: 3.33; 95% CI: 1.68-6.61). However, our investigations did not reveal a robust association between T. gondii infection and other neuropsychiatric conditions, including Alzheimer's disease, dementia, anxiety, depression, neurodegenerative disorders, and peripheral neurological disorders such as neurological and plexus disorders.

Cbl-b negatively regulates TLR/MyD88-mediated anti-Toxoplasma gondii immunity.

IMPORTANCE: This is the first report that a human E3 ubiquitin ligase, Casitas B-lineage lymphoma proto-oncogene B (Cbl-b), functions as a host dependency factor for the intracellular protozoan Toxoplasma gondii and the mechanism for how T. gondii infection inhibits the TLR/MyD88 innate immunity pathway through MyD88 degradation mediated by Cbl-b. This finding is an impactful contribution for understanding the host cell immunity against T. gondii infection.

Anti-infection roles of miR-155-5p packaged in exosomes secreted by dendritic cells infected with Toxoplasma gondii.

BACKGROUND: Toxoplasma gondii is a zoonotic intracellular protozoon that is estimated to infect about 30% of the world's population, resulting in toxoplasmosis in immunocompromised patients and adverse outcomes in cases of primary infection during pregnancy. Exosomes are tubular vesicles secreted by cells, and function in intercellular communication. It has been reported that the exosomes secreted by T. gondii-infected immune cells transmit infection signals to the uninfected cells. However, the mechanism and effect of the exosome transmission are still vague. We therefore investigated the function of the exosomes transmitted from DC2.4 cells infected with the T. gondii RH strain (Tg-DC-Exo) to the uninfected cells, as well as their roles in anti-infection. METHODS: We conducted exosome isolation and identification with ultracentrifugation, transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and western blot (WB) analysis. Exosome uptake by recipient cells was identified by PKH67 assay. The signal transmission and the abundance of miR-155-5p were determined using transwell assay and qRT-PCR. For detection of immune responses, cytokine secretion was evaluated. The T. gondii B1 gene was determined to evaluate tachyzoite proliferation. RESULTS: We observed that Toxoplasma infection upregulated miR-155-5p expression in DC2.4 cell-secreted exosomes, and those exosomes could be ingested by murine macrophage RAW264.7 cells. Tg-DC-Exo and miR-155-5p stimulated host proinflammatory immune responses including increased production of proinflammatory cytokines IL-6 and TNF-α, and proinflammatory marker-inducible nitric oxide synthase (iNOS). The NF-κB pathway was activated by downregulation of SOCS1, leading to inhibition of T. gondii tachyzoite proliferation in RAW264.7 cells. CONCLUSIONS: Our findings provide a novel mechanism for how infected cells transmit infection signals to the uninfected cells through exosome secretion after T. gondii infection, followed by inflammatory responses and anti-infection reactions, which may help us develop a new strategy for toxoplasmosis prevention, especially in immunocompromised patients.

Toxoplasma gondii Type-I ROP18 Targeting Human E3 Ligase TRIM21 for Immune Escape.

Toxoplasma gondii is an intracellular pathogen that exerts its virulence through inhibiting host's innate immune responses, which is mainly related to the type II interferon (IFN-γ) response. IFN-γ inducible tripartite motif 21 (TRIM21), an E3 ligase, plays an important role in anti-infection responses against the intracellular pathogens including bacteria, virus, and parasite. We found that T. gondii virulence factor ROP18 of the type I RH strain (TgROP18I) interacted with human TRIM21, and promoted the latter's phosphorylation, which subsequently accelerated TRIM21 degradation through lysosomal pathway. Furthermore, TRIM21 protein level was found to be upregulated during RH and CEP strains of T. gondii infection. TRIM21 knocking down reduced the ubiquitin labeling on the parasitophorous vacuole membrane (PVM) [which led to parasitophorous vacuole (PV) acidification and death of CEP tachyzoites], and relieved the inhibition of CEP proliferation induced by IFN-γ in human foreskin fibroblast (HFF) cells which was consistent with the result of TRIM21 overexpression. On the other hand, TRIM21 overexpression enhanced the inhibition of CEP proliferation, and inhibited the binding of IκB-α with p65 to activate the IFN-γ-inducible NF-κB pathway, which might be resulted by TRIM21-IκB-α interaction. In brief, our research identified that in human cells, IFN-γ-inducible TRIM21 functioned in the innate immune responses against type III T. gondii infection; however, TgROP18I promoted TRIM21 phosphorylation, leading to TRIM21 degradation for immune escape in type I strain infection.

Strain-specific disruption of interferon-stimulated N-myc and STAT interactor (NMI) function by Toxoplasma gondii type I ROP18 in human cells.

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.

Genome-Wide CRISPR Screen Identifies Host Factors Required by Toxoplasma gondii Infection.

Toxoplasma gondii are obligate intracellular protoza, and due to their small genome and limited encoded proteins, they have to exploit host factors for entry, replication, and dissemination. Such host factors can be defined as host dependency factors (HDFs). Though HDFs are inessential for cell viability, they are critical for pathogen infection, and potential ideal targets for therapeutic intervention. However, information about these HDFs required by T. gondii infection is highly deficient. In this study, the genes of human foreskin fibroblast (HFF) cells were comprehensively edited using the lentiviral CRISPR-Cas9-sgRNA library, and then the lentivirus-treated cells were infected with T. gondii at multiplication of infection 1 (MOI = 1) for 10 days to identify HDFs essential for T. gondii infection. The survival cells were harvested and sent for sgRNA sequencing. The sgRNA sequence matched genes or miRNAs were potential HDFs. Some cells in the lentivirus-treated group could survive longer than those in the untreated control group after T. gondii infection. From a pool of 19,050 human genes and 1,864 human pri-miRNAs, 1,193 potential HDFs were identified, including 1,183 genes and 10 pri-miRNAs (corresponding with 17 mature miRNAs). Among them, seven genes and five mature miRNAs were validated with siRNAs, miRNA inhibitors, and mimics, respectively. Bioinformatics analysis revealed that, among the 1,183 genes, 53 potential HDFs were associated with regulation of host actin cytoskeleton and 23 potential HDFs coded immune negative regulators. This result indicated that actin dynamics were indispensable for T. gondii infection, and some host immune negative regulators may be involved in disarming host defenses. Our findings contribute to the current limited knowledge about host factors required by T. gondii infection and provide us with new targets for medication therapy and vaccine exploitation.

Genome-Wide Bimolecular Fluorescence Complementation-Based Proteomic Analysis of Toxoplasma gondii ROP18's Human Interactome Shows Its Key Role in Regulation of Cell Immunity and Apoptosis.

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.

Characterization of Cytosine Methylation and the DNA Methyltransferases of Toxoplasma gondii.

DNA methylation is a key epigenetic modification which confers phenotypic plasticity and adaptation. Cyst-forming strains of Toxoplasma gondii undergo tachyzoite to bradyzoite conversion after initial acute infection of a host, and the reverse conversion may occur in immune-suppressed hosts. The formation of m5C is catalyzed by DNA methyltransferase (DNMT). We identified two functional DNA methyltransferases, TgDNMTa and TgDNMTb, in T. gondii that may mediate DNA methylation. The recombinant proteins showed intrinsic methyltransferase activity; both have higher transcription levels in bradyzoites than that in tachyzoites. We performed genome-wide analysis of DNA methylation in tachyzoites and bradyzoites. The results showed more methylation sites in bradyzoites than that in tachyzoites. The most significantly enriched GO-terms of genes with DNA methylation were associated with basal cellular processes such as energy metabolism and parasite resistance to host immunity. Tachyzoite proliferation in parasitophorous vacuoles (PV) can be inhibited by the DNA methyltransferase inhibitor 5-azacytidine, a chemical analogue of the nucleotide cytosine that can inactivate DNA methyltransferases. These findings provide the first confirmation of DNA methylation in T. gondii.