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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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.

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.

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