Toxoplasma gondii (T. gondii) is widely spread around the world, which can cause serious harm to immunosuppressed patients. Currently, the commercial test kits are poor at assessing T. gondii infection and vaccine effectiveness, making an urgent need to exploit effective enzyme-linked immunosorbent assay with great performance to compensate for this deficiency. Here, the TgIMP1 recombinant protein was expressed in E. coli BL(21) cells. The TgIMP1 was purified with affinity chromatography and the reactivity was retained with anti-TgIMP1 antibodies. The TgIMP1 was then used to develop an indirect ELISA (IMP1-iELISA) and the reaction conditions of IMP1-iELISA were optimized. As a result, the cut-off value was determined to be 0.2833 by analyzing the OD_450nm values of forty T. gondii-negative sera. The coefficient of variation of 6 T. gondii-positive sera within and between runs were both less than 10%. The IMP1-iELISA was non-cross-reactive with the sera of cytomegalovirus, herpes virus, rubella virus, Cryptosporidium spp., Theileria spp., Neospora spp. and Plasmodium spp.. Furthermore, the sensitivity and specificity of IMP1-iELISA were 98.9% and 96.7%, respectively, based on testing 150 serum samples. The results suggest that this IMP1-iELISA is specific, sensitive, repeatable and can be applied to the detection of T. gondii infections in the medical and health industries.

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 (ATF6β) 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). 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. 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. 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.

Toxoplasma gondii (T. gondii) is a globally distributed protozoan parasite, and its chronic infection has been closely associated with various neuropsychiatric disorders. However, the key susceptibility factors and mechanisms by which T. gondii manipulates host behaviors remain insufficiently understood. In this study, based on the comprehensive behavioral analyses including the AI HomeCage platform, and third-generation Nanopore-seq technology, we demonstrated that the T. gondii strain TgCtwh6 induces depression-like symptoms and cognitive impairments in hosts. These effects are linearly correlated with the expression levels of the host dopamine receptor D2 (DRD2). Furthermore, the behavioral abnormalities can be alleviated by treatment with the DRD2 agonist bromocriptine. Both in vivo and in vitro infection models revealed that the downregulation of DRD2 is significantly correlated with neuroinflammation and neuronal injury in the host central nervous system (CNS). Additionally, our findings suggest that the suppression of DRD2 may be regulated by enhanced RNA m⁶A modification following infection and may function through the DRD2/CRYAB/NF-κB signaling axis. These results emphasized the critical role of DRD2 in mediating T. gondii-induced neuroinflammation and neuropsychiatric abnormalities, providing novel insights into the development of therapeutic strategies for neuropsychiatric disorders associated with parasitic infections.

Background: Toxoplasma gondii can cause severe damage to immunodeficient hosts, and also compromise brain structure and function in immunocompetent hosts during latent infection. In China, the two different isolates, Chinese I (ToxoDB#9) and Chinese III are dominant epidemic strains widely spreading in humans and domestic animals and can lead to latent infection in host brain tissues, but the comparison of their manipulation patterns and mechanisms remains unclear. Methods: Tachyzoites of the TgWh6 (Wh6) strain and the TgCtLHG (LHG) strain were used for establishing in vitro infection models within mouse microglia BV2 cells, and the differences in their invasion and proliferation patterns were observed. C57BL/6 J mice were used to establish in vivo latent infection models. After behavioral tests, the differential expressed transcripts (DETs) of the infected and control animals’ cerebral cortex were sequenced by Nanopore RNA-seq. Functional differences of DETs were analyzed by Gene Ontology enrichment analysis (GO), Kyoto Encyclopedia of Genes and Genomes enrichment analysis (KEGG), and protein–protein interaction (PPI) and cluster analysis. Expression of the key candidates were verified by quantitative polymerase chain reaction (qPCR). Results: In our infection models, we found that Wh6 had more vigorous invasion and proliferation abilities in vitro, while LHG had a greater ability to form cysts in vivo. In the latent infection phase, behavioral changes, including spatial working memory, cognitive and motor abilities, and anxiety, were observed in both Wh6 and LHG infected mice; however, the LHG group showed more serious anxiety. Among DETs, genes related to major histocompatibility complex (MHC) class II molecules were significantly upregulated in the infected mice, while genes related to synaptic transmission and neurodegenerative diseases were downregulated in the infected groups. The downregulated DETs of Sept4, Kcng4, Unc13c, and Prkcg in the WH6 group, which are related to synaptic transmission, and Ndrg2 and Arc in the LHG group, which are related to neurodegenerative diseases, were selected to be the key candidates in the latent infection phase. Conclusions: Compared with WH6, although LHG has a milder invasion ability, it can cause increased behavioral disorders in hosts. Genes related to synaptic transmission and neurodegenerative diseases may be the main causes of host mental and behavioral disorders. Graphical