Background: Rhoptry proteins (ROPs) are secreted effectors that play important roles in the virulence of Toxoplasma gondii by facilitating host cell invasion and immune modulation. Although many ROPs have been predicted, their specific functions remain largely unexplored. This study investigates the roles of 11 previously uncharacterized ROPs in T. gondii biology, with a focus on their contributions to virulence. Methods: Clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated protein 9 (Cas9)–mediated genome editing was employed to generate epitope-tagged and knockout mutants for each candidate ROP in the T. gondii RHΔ ku80 strain. Subcellular localization was determined via immunofluorescence microscopy in both tachyzoite and bradyzoite stages. In vitro assays assessed parasite invasion, replication, egress, and plaque formation. In vivo virulence was evaluated in mouse infection models. To explore molecular mechanisms underlying virulence attenuation, we performed transcriptomic profiling of RHΔ rop64 and RHΔ rop65 knockout strains. Results: All 11 candidate ROPs exhibited rhoptry localization in both tachyzoite and bradyzoite stages. Despite no apparent in vitro growth defects, deletion of ROP64 and ROP65 led to significant attenuation of virulence in mice, with ROP64 showing the most pronounced effect. Transcriptome analysis revealed downregulation of key immune-modulatory genes, including ROP5 , ROP39 , TgIST , and PLP1 . In addition, RHΔ rop64 exhibited broader suppression of ROPs than RHΔ rop65 , suggesting it has a more pronounced role in immune modulation. Conclusions: ROP64 and ROP65 are critical to T. gondii virulence, likely through modulation of the parasite's immune-evasive machinery. Their regulatory influence on effector expression underscores their importance in host adaptation. Importantly, the RHΔ rop64 mutant displays characteristics of an attenuated strain with potential for vaccine development against toxoplasmosis. Graphical

Dense granule protein 47 (GRA47) of Toxoplasma gondii, which localizes to the parasitophorous vacuole membrane (PVM) and the parasitophorous vacuole (PV), is critical for PVM integrity, nutrient permeability, and parasite virulence. However, the global transcriptional response to GRA47 loss remains unexplored. High-throughput RNA sequencing was performed to compare transcriptomic profiles of a GRA47 knockout strain (RHΔgra47) and its parental wild-type RH strain. Differentially expressed genes (DEGs) were identified and analyzed by Gene Ontology (GO) and KEGG enrichment, with selected DEGs validated by reverse transcription quantitative real-time PCR (RT-qPCR). A total of 285 DEGs were identified, comprising 218 upregulated and 67 downregulated genes. Upregulated DEGs were enriched in autophagy, mitophagy, ribosome biogenesis, and GPI anchor biosynthesis pathways, along with nutrient starvation response terms, which may suggest potential roles for the activation of stress response and compensatory metabolic programs. Downregulated DEGs were enriched in host cell entry, rhoptry components, membrane-related terms, and the Hedgehog signaling pathway, a pattern that could suggest a possible association with resource allocation strategy. This first transcriptomic analysis of GRA47 loss in T. gondii identifies transcriptional changes that point to a possible resource allocation strategy prioritizing parasite survival over proliferation and offers a foundational resource for investigating compensatory mechanisms and developing novel intervention strategies. This study reports transcriptional changes only; further experiments are needed to confirm protein-level effects and biological function.

Toxoplasma gondii is an apicomplexan parasite that causes toxoplasmosis, a widespread zoonotic disease leading to serious public health concerns and economic losses to animal husbandry. Currently, highly effective vaccines against toxoplasmosis remain unavailable. This study aimed to investigate the safety, immunogenicity, and protective efficacy of the gra47-deficient mutant strain PruΔgra47 as a live-attenuated vaccine candidate. We evaluated the virulence of PruΔgra47 in a mouse model, determined the optimal immunization dose, and measured serum antibody levels and cytokine profiles. Then, mice immunized with PruΔgra47 were challenged with different T. gondii strains to assess protection against acute and chronic infection. PruΔgra47 displayed significantly attenuated virulence and its ability to form cysts was weakened. Vaccination with 5 × 10⁶ tachyzoites elicited predominantly Th1-skewed immune responses. Immunization with PruΔgra47 provided complete protection against challenge infection with relatively low-virulent PYS strain and homologous Pru strain, prolonged survival against the highly virulent RH strain, and achieved a 90% survival rate with reduced brain cyst burden under chronic challenge. In conclusion, PruΔgra47 is relatively safe and immunogenic in the murine model, and is worth being evaluated in food-producing animals and cats.

Toxoplasma gondii is a widespread intracellular parasite posing serious risks to immunocompromised individuals and pregnant women, and causing significant economic losses to the livestock industry. Current clinical drugs mainly target acute-stage tachyzoites but are ineffective against chronic cysts, necessitating new interventions. The parasite’s sexual stage in the feline intestine remains poorly understood but is critical for transmission. Here, based on our unpublished weighted gene co-expression network analysis of single-cell transcriptomic data of cat intestine after infection with T. gondii, we selected five previously uncharacterized genes, including three CCCH-type zinc finger proteins (TGME49_257130, TGME49_500318, TGME49_313740) and two subtilisin-like proteases (TGME49_248760/sub7, TGME49_235950/sub8). Using the CRISPR-Cas9 system, individual knockout strains were generated from the type II Pru strain. And, we performed phenotypic assessments at this stage as an essential validation to confirm neutrality before feline infection experiments. Phenotypic analyses revealed that none of the knockout strains showed significant differences in vitro growth, replication, egress, in vivo virulence, or cyst formation from the wild-type strain, corroborating the transcriptional prediction. Publicly available ToxoDB transcriptomic data confirmed that four of these genes were highly expressed at the oocyst or feline enteroepithelial stages, but barely expressed in tachyzoites and bradyzoites, consistent with our phenotypic observations. Collectively, these findings indicate that these genes are dispensable for asexual development, while their expression pattern suggests potential involvement in sexual reproduction, a hypothesis that warrants direct experimental validation. This study provides validated knockout strains and candidate genes, establishing an experimental foundation for future functional studies on Toxoplasma sexual development and transmission-blocking strategies.