Xiao-Nan Zheng

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 Here, based on our unpublished weighted gene co-expression network analysis of single-cell transcriptomic data of cat intestine after infection with T. gondii, we selected five previously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.

Toxoplasma gondii is an obligate intracellular apicomplexan parasite that poses serious risks to immunocompromised individuals and the global livestock industry. Our previous study showed that AP2X-1 regulates stage conversion and virulence of T. gondii; however, whether AP2X-1-deficient strains can serve as live-attenuated vaccine candidates is unknown. Here, we evaluated the in vivo virulence, protective efficacy, and induced immunity of the ap2X-1 knockout strain PruΔap2X-1. In Kunming mice, infection with even the highest tested dose (5 × 10⁶ tachyzoites) of PruΔap2X-1 resulted in 100% survival without detectable brain cysts. Vaccination with 10⁶ PruΔap2X-1 tachyzoites conferred complete protection (100% survival) against acute challenge with homologous type II Pru or heterologous ToxoDB#9 PYS strains, whereas control mice exhibited only 0% to 16.7% survival. Moreover, immunized mice orally challenged with Pru cysts showed 100% survival, and no brain cysts were detected under the conditions tested, compared with only 30% survival in control mice challenged with 10 cysts and no survival with 40 cysts. The vaccination induced a Th1-biased response as evidenced by significantly elevated T. gondii-specific total IgG, IgG2a, and the Th1 cytokines IFN-γ and IL-2, along with a significantly reduced IgG1/IgG2a ratio. Collectively, the attenuated PruΔap2X-1 strain generates strong protective immunity against acute and chronic infection with low-virulence strains (the type II Pru and ToxoDB#9 PYS), as well as partial protection against the hypervirulent type I RH strain, positioning it as a potential candidate for a live-attenuated vaccine, with a favorable safety profile observed under the experimental conditions.

Toxoplasmosis, caused by Toxoplasma gondii, remains an important zoonotic disease affecting both human and animal health, particularly in immunocompromised individuals and during pregnancy. Current treatments are unable to eliminate latent tissue cysts and are further limited by adverse effects and prolonged treatment regimens, highlighting the need for effective vaccines. In this study, we evaluated the virulence attenuation and protective efficacy of a CRISPR–Cas9-generated rhoptry protein 64 knockout strain (RHΔrop64) as a live attenuated vaccine candidate in Kunming mice. RHΔrop64 exhibited markedly reduced virulence, and mice tolerated doses of up to 1 × 10² tachyzoites without obvious clinical signs. Immunization provided substantial protection against acute challenge with the parental RHΔku80 and ToxoDB#9 (PYS) strains at 30 days post-vaccination and partial protection against type II (Pru) tachyzoites. In addition, all immunized mice survived following oral challenge with 10 or 40 Pru cysts and exhibited reduced brain cyst burdens among surviving mice. Vaccination also induced measurable humoral and cytokine responses, characterized by increased levels of T. gondii-specific IgG and cytokines that were maintained for at least 75 days post-vaccination. These results suggest that RHΔrop64 has the potential to serve as a live attenuated vaccine candidate, providing substantial, although incomplete, protection against acute challenge with the three T. gondii strains tested and being associated with reduced brain cyst burdens among surviving mice following chronic challenge. Further evaluation is required to determine its safety and applicability as a vaccine candidate for food-producing animals and cats.

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.

Background: Toxoplasma gondii is a globally distributed intracellular parasitic protozoan. It infects nearly all warm-blooded animals and causes a zoonotic disease of worldwide significance. Currently, the only commercially available vaccine, Toxovax ^® , is solely used for the prevention of Toxoplasma -induced abortion in sheep, but it has limitations such as a short shelf life and the potential of reversion to virulence. This study evaluated the safety and immune-protective efficacy of two live-attenuated strains RHΔ tkl1 and PruΔ pp2a-c in sheep. Methods: Sheep were immunized via intramuscular injection in the neck with 1 × 10 ⁷ tachyzoites of RHΔ tkl1 or PruΔ pp2a-c . Sheep were challenged orally with 5 × 10 ⁵ type II Pru oocysts at 28 days post-vaccination (dpv), followed by a second challenge on day 70 with 1 × 10 ⁷ type II Pru tachyzoites injected intramuscularly at 70 dpv. Safety and immuno-protection were evaluated by monitoring clinical symptoms and body temperatures, T. gondii -specific IgG antibody levels, histopathological changes, immunohistochemistry, brain cysts, parasite load, and mouse bioassay results. Results: The results demonstrated that both knockout strains induced only transient fever. Following immunization and subsequent challenge with Pru oocysts, the T. gondii -specific IgG antibody levels in sheep increased rapidly and remained elevated for an extended period. Histopathological analysis indicated mild organ lesions in heart, liver and lung tissues among immunized infected sheep, whereas non-immunized infected sheep exhibited severe widespread inflammation. Immunohistochemical analysis of brain tissue revealed significantly lower values for four parameters (positive cell ratio, density, histochemistry score, immunoreactive score) in immunized groups ( P < 0.01). A significant reduction in brain cysts was observed in immunized and challenged sheep ( P < 0.01) compared with unimmunized and challenged sheep. The parasite burden of T. gondii in heart tissue was significantly reduced ( P < 0.01). Compared with mice inoculated with sheep brain tissue from unimmunized groups challenged with T. gondii Pru oocysts and tachyzoites, mouse bioassay results showed that the mice inoculated with sheep brain tissue from groups immunized with RHΔ tkl1 or PruΔ pp2a-c tachyzoites and subsequently challenged with T. gondii Pru oocysts and tachyzoites exhibited a significantly lower proportion of positive genomic T. gondii DNA in the brain ( P < 0.001), as well as significantly reduced levels of T. gondii -specific antibody IgG in the serum ( P < 0.0001). Similarly, mice inoculated with sheep visceral tissue from the same immunized and challenged groups also showed a significantly reduced proportion of positive genomic T. gondii DNA in the brain ( P < 0.0001) and significantly reduced levels of T. gondii -specific antibody IgG in the serum ( P < 0.0001). Conclusions: The gene knockout strains RHΔ tkl1 and PruΔ pp2a-c showed a certain degree of safety in sheep, and they induced strong humoral and cellular immune responses in sheep, significantly mitigating acute infection symptoms and tissue damage. Notably, PruΔ pp2a-c showed greater potential in suppressing cyst formation. Both strains are potential attenuated candidates against sheep toxoplasmosis.

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.

Background: Toxoplasma gondii is an apicomplexan parasite which infects nearly all warm-blooded animals and humans, causing zoonotic toxoplasmosis. Pork infected with T. gondii is considered a significant source of human infection. Currently, no commercial vaccines are available for porcine toxoplasmosis globally, thus a safe and effective vaccine is urgently needed. This study evaluated the immuno-protective effects of two T. gondii gene knockout attenuated strains, RHΔ tkl1 and PruΔ pp2a-c , in pigs. Methods: Pigs immunized by intramuscular injection with 1 × 10 ⁷ tachyzoites of attenuated RHΔ tkl1 or PruΔ pp2a-c strains were challenged orally with 1.000 sporulated oocysts of the Pru strain at 28 days post-vaccination (dpv), followed by a secondary challenge via intraperitoneal injection of 1 × 10 ⁷ Pru strain tachyzoites at 70 dpv. Clinical signs were monitored. T. gondii -specific antibody levels were examined by enzyme-linked immunosorbent assay. The protective efficacy was evaluated by analyzing pathological lesions, pathological score, parasite load, brain cyst burden, and by mouse bioassay. GraphPad Prism software was employed to perform the log-rank (Mantel Cox) test, Student’s t test and one-way ANOVA. Results: Pigs immunized with either RHΔ tkl1 or PruΔ pp2a-c exhibited only low-grade fever. Combined with pathological changes and pathological scores, these findings support the moderate safety of both strains. Pigs immunized with either RHΔ tkl1 or PruΔ pp2a-c and subsequently challenged with T. gondii Pru oocysts and tachyzoites exhibited a sharp increase in T. gondii -specific antibodies, which remained high for 5 weeks ( P < 0.01). Pathological lesions were alleviated after immunization, with a significant reduction in parasite load observed in tissues including the brain, heart, gastrocnemius, longissimus dorsi, psoas major, and diaphragm ( P < 0.01). Furthermore, a marked decrease in brain cyst burden was recorded ( P < 0.0001). Mouse bioassay results confirmed a significant reduction in the proportion of mouse brain tissues positive for T. gondii genomic DNA in the group immunized and then challenged, compared to the non-immunized challenged group ( P < 0.0001). Conclusions: The two live attenuated RHΔ tkl1 and PruΔ pp2a-c mutants of demonstrated moderate safety, immunogenicity, and protective efficacy in pigs, identifying them as potential candidate vaccines against porcine toxoplasmosis. Graphical

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

Toxoplasma gondii is a globally prevalent parasite capable of establishing lifelong infections, which can have severe consequences in immunocompromised individuals and developing fetuses. GRAs are essential secretory effectors that facilitate nutrient acquisition, modulate host immune responses, and support intracellular survival. In this study, we characterized four putative GRAs (GRA85–88) that co-localize with GRA12 in both tachyzoite and bradyzoite stages. Using CRISPR-Cas9-mediated homologous recombination, we successfully generated knockout strains in both type I RH and type II Pru backgrounds. Phenotypic analysis revealed that GRA85, GRA87, and GRA88 were not individually required for parasite replication, invasion, or virulence. However, deletion of gra86 (PruΔgra86) resulted in a significant reduction in virulence and fewer brain cysts in chronically infected mice, although in vitro growth remained unaffected. Transcriptomic profiling of PruΔgra86 revealed downregulation of bradyzoite–related genes and upregulation of GRAs involved in host interaction. Additionally, in vitro differentiation assays showed impaired bradyzoite development in the absence of GRA86. These findings from murine models and in vitro phenotypic assays highlight GRA86 as a regulator of chronic infection and stage conversion, positioning it as an important player in T. gondii pathogenesis and a promising target for therapeutic intervention.