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Simple Summary: Toxoplasma gondii is a zoonotic pathogenic apicomplexan parasite that infects approximately one third of the global population. In immunosuppressed individuals, the infection may be fatal. Current anti-toxoplasmosis drugs have low efficacy and cause side effects, and no vaccine is available to prevent human toxoplasmosis. To search for potential virulence genes of T. gondii with a view to serving as new drug or vaccine targets for toxoplasmosis, we selected 11 genes of interest (GOIs), which were tentative microneme proteins (MICs) with unknown functions, and applied the CRISPR-Cas9 system to construct 11 epitope tagging strains and gene knockout strains to explore their intracellular localization and biological functions. The results showed that nine tentative MICs were localized or partially localized in the microneme, one GOI was localized in endoplasmic reticulum, and another GOI was localized in the dense granule. Deletion of the 11 genes showed no significant effect on the in vitro growth and virulence of the tachyzoites of T. gondii type I RH strain. This study investigated the role of the 11 tentative mics in T. gondii tachyzoites, which expanded the knowledge of the functionally unknown genes of T. gondii. In the future, roles of these genes in other life stages and other genotypes of T. gondii will be explored. Toxoplasma gondii, a pathogenic apicomplexan parasite, infects approximately one third of the world's population and poses a serious threat to global public health. Microneme proteins (MICs) secreted by the microneme, an apical secretory organelle of T. gondii, play important roles in the invasion, motility, and intracellular survival of T. gondii. In this study, we selected 11 genes of interest (GOIs) of T. gondii, tentative MICs predicted to be localized in micronemes, and we used the CRISPR-Cas9 system to construct epitope tagging strains and gene knockout strains to explore the localization and function of these 11 tentative MICs. Immunofluorescence assay showed that nine tentative MICs (TGME49_243930, TGME49_200270, TGME49_273320, TGME49_287040, TGME49_261710, TGME49_205680, TGME49_304490, TGME49_245485, and TGME49_224620) were localized or partially localized in the microneme, consistent with the prediction. However, TGME49_272380 and TGME49_243790 showed different localizations from the prediction, being localized in the endoplasmic reticulum and the dense granule, respectively. Further functional characterization of the 11 RHΔGOI strains revealed that deletion of these 11 GOIs had no significant effect on plaque formation, intracellular replication, egress, invasion ability, and virulence of T. gondii. Although these 11 GOIs are not essential genes for the growth and virulence of tachyzoites of type I RH strain, they may have potential roles in other developmental stages or other genotypes of T. gondii. Thus, further research should be performed to explore the possible role of the nine mics and the other two GOIs in other life cycle stages and other genotypes of T. gondii. [ABSTRACT FROM AUTHOR]

Eukaryotic translation initiation factors (eIFs) are crucial for initiating protein translation and ensuring the correct assembly of mRNA-ribosomal subunit complexes. In this study, we investigated the effects of deleting six eIFs in the apicomplexan parasite Toxoplasma gondii using the CRISPR-Cas9 system. We determined the subcellular localization of these eIFs using C-terminal endogenous tagging and immunofluorescence analysis. Four eIFs (RH::315150-6HA, RH::286090-6HA, RH::249370-6HA, and RH::211410-6HA) were localized in the cytoplasm, while RH::224235-6HA was localized in the apicoplast. Additionally, RH::272640-6HA was found in both the basal complex and the cytoplasm of T. gondii. Functional characterization of the six RHΔeIFs strains was conducted using plaque assay, cell invasion assay, intracellular growth assay and egress assay in vitro, and virulence assay in mice. Disruption of five eIF genes (RHΔ315150, RHΔ272640, RHΔ249370, RHΔ211410, and RHΔ224235) did not affect the ability of the T. gondii RH strain to invade, replicate, form plaques and egress in vitro, or virulence in Kunming mice (p > 0.05). However, the RHΔ286090 strain showed slightly reduced invasion efficiency and virulence (p < 0.01) compared to the other five RHΔeIFs strains and the wild-type strain. The disruption of the TGGT1_286090 gene significantly impaired the ability of tachyzoites to differentiate into bradyzoites in both type I RH and type II Pru strains. These findings reveal that the eukaryotic translation initiation factor TGGT1_286090 is crucial for T. gondii bradyzoite differentiation and may serve as a potential target for drug development and an attenuated vaccine against T. gondii. [ABSTRACT FROM AUTHOR]

Toxoplasmosis caused by Toxoplasma gondii is an important zoonosis of human and animal health significance. Current chemical therapeutics have side effects, and no commercially available vaccine is licensed for the prevention of toxoplasmosis in humans and most animals. Developing a safe and effective vaccine with long-term protection against T. gondii infection is necessary to control toxoplasmosis. HAD2a is a key member of the haloacid dehalogenase (HAD) phosphatase family, which is essential for T. gondii daughter budding. However, the role of HAD2a in T. gondii virulence remains unknown. In this study, we successfully constructed the had2a gene knockout strain in the T. gondii-type I RH strain (RHΔhad2a) and determined its role in virulence and vaccination. These results demonstrate that HAD2a played an important role in parasite daughter budding and in vitro replication. Knockout of the had2a gene attenuated the virulence of the T. gondii-type I RH strain. Vaccination with RHΔhad2a tachyzoites induced a Th1-biased immune response, provided partial protection against acute T. gondii infection in mice by highly virulent tachyzoites of RH and PYS (ToxoDB#9, Chinese I) strains, and conferred strong protection against challenge infection by cysts and oocysts of the less virulent type II Pru strain. These results demonstrate that T. gondii had2a is important for its in vitro proliferation and virulence in mice and that RHΔhad2a may be used as a candidate strain to generate a multiple gene knockout live-attenuated strain or be collaboratively applied with other live-attenuated strains to confer more effective protection against T. gondii infection. [ABSTRACT FROM AUTHOR]

Sexual development in Toxoplasma gondii is a multistep process that culminates in the production of oocysts, constituting approximately 50% of human infections. However, the molecular mechanisms governing sexual commitment in this parasite remain poorly understood. Here, we demonstrate that the transcription factors AP2XI-2 and AP2XII-1 act as negative regulators, suppressing merozoite-primed pre-sexual commitment during asexual development. Depletion of AP2XI-2 in type II Pru strain induces merogony and production of mature merozoites in an alkaline medium but not in a neutral medium. In contrast, AP2XII-1-depleted Pru strain undergoes several rounds of merogony and produces merozoites in a neutral medium, with more pronounced effects observed under alkaline conditions. Additionally, we identified two additional AP2XI-2-interacting proteins involved in repressing merozoite programming. These findings underscore the intricate regulation of pre-sexual commitment by a network of factors and suggest that AP2XI-2 or AP2XII-1-depleted Pru parasites can serve as a model for studying merogony in vitro. Wang et al. discovered that AP2XI-2 and AP2XII-1 negatively regulate merozoite-primed pre-sexual commitment in Toxoplasma gondii, and parasites depleted of either AP2XI-2 or AP2XII-1 can serve as a valuable in vitro model for studying merogony. [ABSTRACT FROM AUTHOR]

The Zinc finger protein (ZFP) family is widely distributed in eukaryotes and interacts with DNA, RNA, and various proteins to participate in many molecular processes. In the present study, the biological functions of eight ZFP genes in the lytic cycle and the pathogenicity of Toxoplasma gondii were examined using the CRISPR-Cas9 system. Immunofluorescence showed that four ZFPs (RH248270-HA, RH255310-HA, RH309200-HA, and RH236640-HA) were localized in the cytoplasm, and one ZFP (RH273150-HA) was located in the nucleus, while the expression level of RH285190-HA, RH260870-HA, and RH248450-HA was undetectable. No significant differences were detected between seven RHΔzfp strains (RHΔ285190, RHΔ248270, RHΔ260870, RHΔ255310, RHΔ309200, RHΔ248450, and RHΔ236640) and the wild-type (WT) strain in the T. gondii lytic cycle, including plaque formation, invasion, intracellular replication, and egress, as well as in vitro virulence (p > 0.05). However, the RHΔ273150 strain exhibited significantly lower replication efficiency compared to the other seven RHΔzfp strains and the WT strain, while in vivo virulence in mice was not significantly affected. Comparative expression analysis of the eight zfp genes indicates that certain genes may have essential functions in the sexual reproductive stage of T. gondii. Taken together, these findings expand our current understanding of the roles of ZFPs in T. gondii. [ABSTRACT FROM AUTHOR]

Toxoplasma gondii infection is prevalent in humans and animals worldwide. In this study, recombinant eukaryotic expression plasmids (pVAX-GRA24, pVAX-GRA25 and pVAX-MIC6) were constructed, and then injected into Kunming mice intramuscularly, as cocktailed plasmids or as single-gene plasmids. We evaluated immune protective responses by detecting the titer of antibodies and cytokine production of IFN-γ, IL-2, IL-4, IL-10, IL-12 and IL-23, the percentages of the subclasses of T lymphocytes, as well as the records of the survival time and cyst decrement in the brain of the mouse model after challenge with the T. gondii RH and Pru strains, respectively. Compared with the control groups, antibody and cytokine production were significantly increased, while the survival times of mice in all immunized groups were also prolonged, and the number of T. gondii cysts in their brains were decreased significantly (29.03% for pVAX-GRA24; 40.88% for pVAX-GRA25; 37.70% for pVAX-MIC6; 48.06% for pVAX-GRA24 + pVAX-GRA25; and 55.37% for pVAX-GRA24 + pVAX-GRA25 + pVAX-MIC6). The mouse group immunized with the three-gene cocktail (TgGRA24 + TgGRA25 + TgMIC6) had better performance in each detection index than the mouse groups immunized with the two-gene cocktail of TgGRA24 + TgGRA25, which was better than that in the group immunized with the single gene vaccine of TgGRA24, TgMIC6 or TgGRA25. In conclusion, TgGRA24 or TgGRA25 may be good vaccine candidates against T. gondii infection, but the three-gene cocktail of TgGRA24, TgMIC6 and TgGRA25 may induce the strongest protective immunity. Further studies of multi-antigenic DNA vaccines or cocktailed vaccines against T. gondii infection are necessary.

Toxoplasma gondii causes one of the most common protozoal diseases of humans and animals worldwide. With the aim of designing an effective vaccine against T. gondii infection, we examined the immunogenicity of a DNA vaccine expressing heat shock protein 40 (HSP40) against challenge with T. gondii (type I RH and type II Pru) strains in Kunming mice. The plasmid pVAX1-HSP40 was constructed and used to immunize mice by intramuscular injection for three sequential immunizations with two-week intervals. This immunization regimen significantly reduced parasite cyst burden in pVAX1-HSP40-immunized mice (1871.9 ± 142.3) compared with control mouse groups immunized with pVAX1 (3479.2 ± 204.4), phosphate buffered saline (3024.4 ± 212.8), or left untreated (3275.0 ± 179.8) as healthy controls (p

Phenotypic switching between tachyzoite and bradyzoite is the fundamental mechanism underpinning the pathogenicity and adaptability of the protozoan parasite Toxoplasma gondii. Although accumulation of cytoplasmic starch granules is a hallmark of the quiescent bradyzoite stage, the regulatory factors and mechanisms contributing to amylopectin storage in bradyzoites are incompletely known. Here, we show that T. gondii protein phosphatase 2A (PP2A) holoenzyme is composed of a catalytic subunit PP2A-C, a scaffold subunit PP2A-A and a regulatory subunit PP2A-B. Disruption of any of these subunits increased starch accumulation and blocked the tachyzoite-to-bradyzoite differentiation. PP2A contributes to the regulation of amylopectin metabolism via dephosphorylation of calcium-dependent protein kinase 2 at S679. Phosphoproteomics identified several putative PP2A holoenzyme substrates that are involved in bradyzoite differentiation. Our findings provide novel insight into the role of PP2A as a key regulator of starch metabolism and bradyzoite differentiation in T. gondii. Protein phosphorylation and dephosphorylation are essential aspects of biology. Wang et al., report that Toxoplasmagondiiprotein phosphatase 2A (PP2A) mediated dephosphorylation is critical for starch metabolism and bradyzoite differentiation. [ABSTRACT FROM AUTHOR]

Toxoplasma gondii, an intracellular zoonotic protozoan parasite, is possibly the most widespread parasite of warm-blooded animals and can cause serious public health problems and economic losses worldwide. TgCDPK2, a member of the T. gondii calcium-dependent protein kinase family, was recently identified as an essential regulator for viable cyst development in T. gondii. In the present study, we evaluated the protective immunity induced by DNA vaccination based on a recombinant eukaryotic plasmid, pVAX-TgCDPK2, against acute toxoplasmosis in mice. BALB/c mice were intramuscularly immunized with pVAX-TgCDPK2 plasmid and then challenged by infection with the highly virulent RH strain of T. gondii. The specific immune responses and protective efficacy against T. gondii were analyzed by cytokine and serum antibody measurements, lymphocyte proliferation assays, flow cytometric on lymphocytes and the survival time of mice after challenge. Our results showed that mice immunized with pVAX-TgCDPK2 could elicit special humoral and cellular responses, with higher levels of IgG antibody, and increased levels of Th1-type cytokines IFN-γ, IL-12(p70), and CD3 + CD4 + CD8 − and CD3 + CD8 + CD4 − T cells, and had a prolonged survival time (14.0 ± 2.32 days) compared to control mice. These results demonstrate that pVAX-TgCDPK2 is a potential vaccine candidate against acute toxoplasmosis.

Lysine malonylation is a post-translational modification (PTM), which regulates many cellular processes. Limited information is available about the level of lysine malonylation variations between Toxoplasma gondii strains of distinct genetic lineages. Yet, insights in such variations are needed to understand the extent to which lysine malonylation contributes to the differences in the virulence and repertoire of virulence factors between T. gondii genotypes. In this study, we profiled lysine malonylation in T. gondii using quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS) and immuno-affinity purification. This analysis was performed on three T. gondii strains with distinctive pathogenicity in mice, including RH strain (type I), PRU strain (type II), and VEG strain (type III). In total, 111 differentially malonylated proteins and 152 sites were upregulated, and 17 proteins and 17 sites were downregulated in RH strain versus PRU strain; 50 proteins and 59 sites were upregulated, 50 proteins and 53 sites were downregulated in RH strain versus VEG strain; and 72 proteins and 90 sites were upregulated, and 7 proteins and 8 sites were downregulated in VEG strain versus PRU strain. Differentially malonylated proteins were involved in key processes, such as those mediating the regulation of protein metabolism, stress response, glycolysis, and actin cytoskeleton. These results reveal an association between lysine malonylation and intra-species virulence differences in T. gondii and offer a new resource for elucidating the contribution of lysine malonylation to energy metabolism and virulence in T. gondii. Author summary: Lysine malonylation has been shown to play important roles in various biological processes in Toxoplasma gondii. Here, we used quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS) and immuno-affinity purification to test the hypothesis that lysine malonylation underpins the inter-genotype differences in the virulence of T. gondii. Several up-regulated and down- regulated malonylated proteins were identified in the tachyzoites of RH (type I) strain, PRU (type II) strain, and VEG (type III) strain. Differentially regulated malonylated proteins were enriched in many biological and metabolic pathways, and were found to contribute T. gondii energy metabolism, stress response, and infectivity, suggesting the role of lysine malonylation in the regulation of T. gondii virulence. These findings expand our knowledge of lysine malonylation in T. gondii and provide more insight into the mechanisms mediating the virulence differences between T. gondii strains of different genotypes. [ABSTRACT FROM AUTHOR]

Toxoplasma gondii is a worldwide distributed zoonotic pathogen that threatens public health. However, there have been limited data for T. gondii infection in wild rats (Rattus norvegicus) in China. In the present study, a total of 227 wild rats were captured from three mink farms to investigate T. gondii infection in Shandong Province, eastern China. The DNA was extracted from 25 mg rats' brain tissues and subjected to a PCR amplification by targeting to the T. gondii B1. In 227 wild rat samples, 18 samples (7.93%) were positive for T. gondii. Then, the positive samples were further genotyped based on eight genetic markers, including eight nuclear loci (SAG1, 5′-SAG2, and 3′-SAG2, alternative SAG2, SAG3, GRA6, c29–2, and L358) and an apicoplast locus (Apico) by using the multilocus PCR-restriction fragment length polymorphism technology. Of these samples, eight were genotyped at nine nuclear loci, and two were genotyped at eight nuclear loci, forming three known genotypes (ToxoDB no. 43, ToxoDB no. 91, and ToxoDB no. 189) and two new genotypes. The closest ToxoDB genotypes were observed in wild rats, suggesting the differences in the population structure of the T. gondii between breed farm animals and wild rats. These data revealed the genetic variability of T. gondii in wild rats on mink farms in Shandong Province, with possible implication for public health. [ABSTRACT FROM AUTHOR]

Abstract Background Toxoplasma gondii is a highly prevalent protozoan parasite infecting a wide range of animals and humans. The epidemiological and biological diversity of T. gondii has resulted in a high genetic variation and unusual population structure in this parasite. This study examined sequence diversity in dense granule 5 (GRA5) gene among T. gondii isolates from different hosts and geographical regions. Methods The entire genome region of the GRA5 gene was amplified and sequenced from 14 T. gondii isolates, and phylogenetic relationship among these T. gondii isolates was reconstructed using Bayesian inference (BI) and maximum parsimony (MP) based on the GRA5 sequences. Results The complete sequence of the GRA5 gene was 1614 bp in length for strains TgCatBr5 and MAS, but 1617 bp for the other 12 strains. Sequence analysis identified 41 (0–1.7%) variable nucleotide positions among all isolates, with 18 variations of these being in the coding region. Variable positions in the coding region resulted in 11 amino acid substitutions, and a deletion of 3 bp in the strains TgCatBr5 and MAS leading to the deletion of one amino acid. Sequence variations resulted in the existence of polymorphic restriction sites for endonucleases AatII and MluI, allowing the differentiation of the three major clonal lineage types I, II and III by PCR-RFLP. Phylogenetic analyses using BI and MP supported the clear differentiation of the examined T. gondii strains into their respective genotypes. Conclusions This study demonstrated the existence of sequence variability in the GRA5 gene sequence among T. gondii isolates from different hosts and geographical regions, which allowed the differentiation of the examined T. gondii strains into their respective genotypes, suggesting that this highly polymorphic GRA5 locus may provide a new genetic marker for population genetic studies of T. gondii isolates.

Toxoplasma gondii is an obligate intracellular protozoan parasite, which has a worldwide distribution and can infect a large number of warm-blooded animals and humans. T. gondii must colonize and proliferate inside the host cells in order to maintain its own survival by securing essential nutrients for the development of the newly generated tachyzoites. The type II fatty acid biosynthesis pathway (FASII) in the apicoplast is essential for the growth and survival of T. gondii. We investigated whether deletion of genes in the FASII pathway influences the in vitro growth and in vivo virulence of T. gondii. We focused on beta-hydroxyacyl-acyl carrier protein dehydratase (FabZ) and oxidoreductase, short chain dehydrogenase/reductase family proteins ODSCI and ODSCII. We constructed T. gondii strains deficient in FabZ, ODSCI , and ODSCII using CRISPR-Cas9 gene editing technology. The results of immunofluorescence assay, plaque assay, proliferation assay and egress assay showed that in RHΔ FabZ strain the apicoplast was partly lost and the growth ability of the parasite in vitro was significantly inhibited, while for RHΔ ODSCI and RHΔ ODSCII mutant strains no similar changes were detected. RHΔ FabZ exhibited reduced virulence for mice compared with RHΔ ODSCI and RHΔ ODSCII , as shown by the improved survival rate. Deletion of FabZ in the PRU strain significantly decreased the brain cyst burden in mice compared with PRUΔ ODSCI and PRUΔ ODSCII. Collectively, these findings suggest that FabZ contributes to the growth and virulence of T. gondii , while ODSCI and ODSCII do not contribute to these traits. [ABSTRACT FROM AUTHOR]

Toxoplasma gondii is an obligate intracellular parasite capable of establishing persistent infection within the host brain and inducing severe neuropathology. Peptides are important native molecules responsible for a wide range of biological functions within the central nervous system. However, peptidome profiling in host brain during T. gondii infection has never been investigated. Using a label-free peptidomics approach (LC–MS/MS), we identified a total of 2,735 endogenous peptides from acutely infected, chronically infected and control brain samples following T. gondii infection. Quantitative analysis revealed 478 and 344 significantly differentially expressed peptides (DEPs) in the acute and chronic infection stages, respectively. Functional analysis of DEPs by Gene Ontology suggested these DEPs mainly originated from cell part and took part in cellular process. We also identified three novel neuropeptides derived from the precursor protein cholecystokinin. These results demonstrated the usefulness of quantitative peptidomics in determining bioactive peptides and elucidating their functions in the regulation of behavior modification during T. gondii infection. [ABSTRACT FROM AUTHOR]

Abstract Background Toxoplasmosis, caused by an obligate intracellular protozoan parasite Toxoplasma gondii, has been a serious clinical and veterinary problem. Effective DNA vaccines against T. gondii can prevent and control the spread of toxoplasmosis, which is important for both human health and the farming industry. The T. gondii 14-3-3 protein has been proved to be antigenic and immunogenic and was a potential vaccine candidate against toxoplasmosis. In this study, we evaluated the immune responses induced by recombinant plasmids encoding T. gondii surface antigen 1 (SAG1) and 14-3-3 protein by immunizing BALB/c mice intramuscularly. Methods In the present study, BALB/c mice were randomly divided into five groups, including three experimental groups (pSAG1, p14-3-3 and pSAG1/14-3-3) and two control groups (PBS and pBudCE4.1), and were immunized intramuscularly three times. The levels of IgG antibodies and cytokine production in mouse sera were determined by enzyme-linked immunosorbent assays (ELISA). Two weeks after the last immunization, all mice were challenged intraperitoneally (i.p.) with 1×104 tachyzoites of T. gondii and the survival time of mice was observed and recorded every day. Results Mice vaccinated with pSAG1, p14-3-3 or pSAG1/14-3-3 developed high levels of IgG2a and gamma interferon (IFN-γ) and low levels of interleukin-4 (IL-4) and interleukin-10 (IL-10) compared to control groups (PBS or pBudCE4.1), which suggested a modulated Th1 type immune response (P4 tachyzoites of T. gondii (RH strain), the survival time of mice in experimental groups was longer than control groups (P Conclusions The study suggested that T. gondii 14-3-3 protein can induce effective immune responses in BALB/c mice and was a novel DNA vaccine candidate against toxoplasmosis, and the immune protective efficacy elicited by SAG1 gene was also demonstrated. Our results also showed multi-gene vaccine significantly enhanced immune responses and protective efficacy and was superior to the single-gene vaccine.

Abstract Background Toxoplasmosis is an important parasitic zoonosis caused by the protozoan Toxoplasma gondii that is distributed world-wide and infects a variety of hosts. However, the prevalence of T. gondii in the environment (such as soil, water and food) is largely unknown. Due to the technical difficulty in oocyst counting directly, an alternative assay using the serologic status of T. gondii in free-living animals, such as stray or free-living dogs, as an indicator, can be used to evaluate environmental contamination indirectly, as they are exposed to the same risk of infection as humans and other animals. Results In the present study, 231 stray or free-living dogs across an urban-rural gradient were examined to assess the frequency of T. gondii in the environment. Specific antibodies to T. gondii were found in 93 dogs (40.3%) by enzyme-linked immunosorbent assay (ELISA), and no statistically significant differences were observed in seroprevalences of T. gondii between urban dogs (38.7%) and rural dogs (41%) (p > 0.05). Conclusions A high seroprevalence of T. gondii in stray or free-living dogs in the present study indicates that there would be a wide distribution and a constant infection pressure of T. gondii across an urban-rural gradient, and the oocysts of T. gondii in the environment would be an important source of infection for humans and other animals both in urban and rural areas in China.

Abstract Toxoplasmosis is a zoonotic infection of humans and animals, caused by the opportunistic protozoan Toxoplasma gondii, a parasite belonging to the phylum Apicomplexa. Infection in pregnant women may lead to abortion, stillbirth or other serious consequences in newborns. Infection in immunocompromised patients can be fatal if not treated. On average, one third of people are chronically infected worldwide. Although very limited information from China has been published in the English journals, T. gondii infection is actually a significant human health problem in China. In the present article, we reviewed the clinical features, transmission, prevalence of T. gondii infection in humans in China, and summarized genetic characterizations of reported T. gondii isolates. Educating the public about the risks associated with unhealthy food and life style habits, tracking serological examinations to special populations, and measures to strengthen food and occupational safety are discussed.