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The analysis of the subcellular localization and function of dense granule proteins (GRAs) is of central importance for the understanding of host-parasite interaction and pathogenesis of Toxoplasma gondii infection. Here, we identified 15 novel GRAs and used C-terminal endogenous gene tagging to determine their localization at the intravacuolar network (IVN), parasitophorous vacuole (PV), or PV membrane (PVM) in the tachyzoites and at the periphery of the bradyzoites-containing cysts. The functions of the 15 gra genes were examined in type I RH strain and 5 of these gra genes were also evaluated in the cyst-forming type II Pru strain. The 15 novel gra genes were successfully disrupted by using CRISPR-Cas9 mediated homologous recombination and the results showed that 13 gra genes were not individually essential for T. gondii replication in vitro or virulence in mice during acute and chronic infection. Intriguingly, deletion of TGME49_266410 and TGME49_315910 in both RH and Pru strains decreased the parasite replication in vitro and attenuated its virulence, and also reduced the cyst-forming ability of the Pru strain in mice during chronic infection. Comparison of the transcriptomic profiles of the 15 gra genes suggests that they may play roles in other life cycle stages and genotypes of T. gondii. Taken together, our findings improve the understanding of T. gondii pathogenesis and demonstrate the involvement of two novel GRAs, TGME49_266410 and TGME49_315910, in the parasite replication and virulence. IMPORTANCE Dense granule proteins (GRAs) play important roles in Toxoplasma gondii pathogenicity. However, the functions of many putative GRAs have not been elucidated. Here, we found that 15 novel GRAs are secreted into intravacuolar network (IVN), parasitophorous vacuole (PV), or PV membrane (PVM) in tachyzoites and are located at the periphery of the bradyzoite-containing cysts. TGME49_266410 and TGME49_315910 were crucial to the growth of RH and Pru strains in vitro . Deletion of TGME49_266410 and TGME49_315910 attenuated the parasite virulence in mice. However, disruption of other 13 gra genes did not have a significant impact on the proliferation and pathogenicity of T. gondii in vitro or in vivo . The marked effects of the two novel GRAs (TGME49_266410 and TGME49_315910) on the in vitro growth and virulence of T. gondii are notable and warrant further elucidation of the temporal and spatial dynamics of translocation of these two novel GRAs and how do they interfere with host cell functions.

Dense granule protein 12 (GRA12) is implicated in a range of processes related to the establishment of Toxoplasma gondii infection, such as the formation of the intravacuolar network (IVN) within the parasitophorous vacuole (PV). This protein is also thought to be important for T. gondii-host interaction, pathogenesis, and immune evasion, but their exact roles remain unknown. In this study, the contributions of GRA12 to the molecular pathogenesis of T. gondii infection were examined in vitro and in vivo. Deletion of GRA12 in type I RH and type II Pru T. gondii strains did not affect the parasite growth and replication in vitro, however, it caused a significant reduction in the parasite virulence and tissue cyst burden in vivo. T. gondii Δgra12 mutants were more vulnerable to be eliminated by host immunity, without the accumulation of immunity-related GTPase a6 (Irga6) onto the PV membrane. The ultrastructure of IVN in Δgra12 mutants appeared normal, suggesting that GRA12 is not required for biogenesis of the IVN. Combined deletion of GRA12 and ROP18 induced more severe attenuation of virulence compared to single Δgra12 or Δrop18 mutant strains. These data suggest a functional association between GRA12 and ROP18 that is revealed by the severe attenuation of virulence in a double mutant relative to the single individual mutations. Future studies are needed to define the molecular basis of this putative association. Collectively these findings indicate that although GRA12 is not essential for the parasite growth and replication in vitro, it contributes to the virulence and growth of T. gondii in mice., (© 2020 Federation of American Societies for Experimental Biology.)

In this study, we characterized the role of amylo-alpha-1,6-glucosidase (Aa16GL) in the biology and infectivity of Toxoplasma gondii , using Aa16GL-deficient parasites of type I RH and type II Prugniaud (Pru) strains. The subcellular localization of Aa16GL protein was characterized by tagging a 3 × HA to the 3' end of the Aa16GL gene endogenous locus. Immunostaining of the expressed Aa16GL protein revealed that it is located in several small cytoplasmic puncta. Functional characterization of ΔAa16GL mutants using plaque assay, egress assay and intracellular replication assay showed that parasites lacking Aa16GL exhibit a slight reduction in the growth rate, but remained virulent to mice. Although PruΔAa16GL tachyzoites retained the ability to differentiate into bradyzoites in vitro , they exhibited slight reduction in their ability to form cysts in mice. These findings reveal new properties of Aa16GL and suggest that while it does not have a substantial role in mediating T. gondii infectivity, this protein can influence the formation of parasite cysts in mice., (Copyright © 2019 Cao, Wang, Elsheikha, Li, Sun, Liang, Zhang and Lin.)

We characterized the porcine tissue transcriptional landscapes that follow Toxoplasma gondii infection. RNAs were isolated from liver, spleen, cerebral cortex, lung, and mesenteric lymph nodes (MLNs) of T. gondii -infected and uninfected (control) pigs at days 6 and 18 postinfection, and were analyzed using next-generation sequencing (RNA-seq). T. gondii altered the expression of 178, 476, 199, 201, and 362 transcripts at 6 dpi and 217, 223, 347, 119, and 161 at 18 dpi in the infected brain, liver, lung, MLNs and spleen, respectively. The differentially expressed transcripts (DETs) were grouped into five expression patterns and 10 sub-clusters. Gene Ontology enrichment and pathway analysis revealed that immune-related genes dominated the overall transcriptomic signature and that metabolic processes, such as steroid biosynthesis, and metabolism of lipid and carboxylic acid, were downregulated in infected tissues. Co-expression network analysis identified transcriptional modules associated with host immune response to infection. These findings not only show how T. gondii infection alters porcine transcriptome in a tissue-specific manner, but also offer a gateway for testing new hypotheses regarding human response to T. gondii infection.

Important progress has been made in understanding how immunity is elicited against Toxoplasma gondii - a complex pathogen with multiple mechanisms of immune evasion. Many vaccine candidates have been tested using various strategies in animal models. However, none of these strategies has delivered as yet, and important challenges remain in the development of vaccines that can eliminate the tissue cysts and/or fully block vertical transmission. In this review, we provide an overview of the current understanding of the host immune response to T. gondii infection and summarize the key limitations for the development of an effective, safe, and durable toxoplasmosis vaccine. We also discuss how the successes and failures in developing and testing vaccine candidates have provided a roadmap for future vaccine development., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Background: The threat of Toxoplasma gondii infection in immunocompromised individuals and pregnant women necessitates the development of a safe and effective vaccine. Here, we examined the immune protection conferred by a live attenuated strain of T. gondii., Methods: We tested the efficacy of intraperitoneal vaccination using 500 Ca2+-dependent protein kinase 2 (cdpk2)-deficient tachyzoites of T. gondii Pru strain against acute, chronic, and congenital toxoplasmosis in mice. The kinetics of antibody response, cytokines, and other quantifiable correlates of protection against T. gondii infection were determined., Results: Vaccination with Pru:Δcdpk2 induced a high level of anti-T. gondii immunoglobulin G titer, type 1 T-helper (Th1) response at 28 days postvaccination, and a mixed Th1/type 2 T-helper response at 70 days postvaccination. All vaccinated mice survived a heterologous challenge with 1000 tachyzoites of RH or ToxoDB#9 (PYS or TgC7) strains. Also, vaccination protected against homologous infection with 20 T. gondii Pru cysts, and improved pregnancy outcome by reducing parasite cyst load in the brain, maintaining litter size and body weight of pups born to vaccinated dams challenged with 10 Pru cysts compared to pups born to unvaccinated dams., Conclusions: The use of T. gondii Pru:Δcdpk2 mutant strain represents a promising approach to protection against acute, chronic, and congenital toxoplasmosis in mice.

Toxoplasma gondii is an obligatory intracellular protozoan, which infects most of the warm-blooded animals, causing serious public health problems and enormous economic losses worldwide. The rhoptry effector protein 54 (ROP54) has been indicated as a virulence factor that promotes Toxoplasma infection by modulating GBP2 loading onto parasite-containing vacuoles, which can modulate some aspects of the host immune response. In order to evaluate the immuno-protective value of ROP54, we constructed a eukaryotic recombinant plasmid expressing T. gondii ROP54 and intramuscularly immunized Kunming mice with this recombinant plasmid against acute and chronic toxoplasmosis. All mice immunized with pVAX-ROP54 elicited a high level of specific antibody responses, a significant increase of lymphocyte proliferation, and a significant level of Th1-type cytokines (IFN-γ, IL-2 and IL-12p70), in addition to an increased production of Th2-type cytokines (IL-4 and IL-10). These results demonstrated that pVAX-ROP54 induced significant cellular and humoral (Th1/Th2) immune responses, which extended the survival time (13.0±1.15days for pVAX-ROP54 vs 6.7±0.48days for pVAX I, 6.8±0.42days for PBS and 6.5±0.53 for blank control) and significantly reduced cyst burden (35.9% for pVAX-ROP54, 1% for pVAX I and 2% for PBS, compared with blank control) of immunized mice. These results indicate that the recombinant ROP54 plasmid can provide partial protection and might be a potential vaccine candidate against acute and chronic toxoplasmosis., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Toxoplasma gondii is a worldwide prevalent pathogen that infects most of the warm-blood vertebrates. To investigate the regulation network of splenic miRNAs altered by acute infection with T. gondii, we herein investigated the changes of miRNA profile in mouse spleen via next generation sequencing and bioinformatics analysis. A total of 379 miRNAs was identified, 131 miRNAs of them were differentially expressed (including 97 upregulated and 34 downregulated miRNAs). 48 differentially expressed miRNAs had validated targets in the miRWalk2.0 database. Gene Ontology (GO) enrichment analysis revealed that the validated targets of differently expressed miRNAs were significantly enriched in gene transcription regulation. It suggested that T. gondii can modulate host gene expression through targeting to trans-regulation factors. The genes involved in apoptosis or anti-apoptosis were both targeted by differentially expressed miRNAs. The change of power balance between the miRNAs targeting host apoptosis genes and those regulating host anti-apoptosis genes contributes to the fate of host apoptosis process. Twelve pathways were significantly enriched in KEGG analysis with most of them being cancer related, including pathways in cancer, pancreatic cancer, colorectal cancer, axon guidance, MAPK signaling pathway, focal adhesion, chronic myeloid leukemia, renal cell carcinoma, prostate cancer, glioma, regulation of actin cytoskeleton, and Wnt signaling pathway. Our study showed a changed miRNA regulation network in mouse spleen infected by T. gondii. These findings will be helpful for better understanding of miRNA regulation network in host-T. gondii interaction, revealing the relationship among T. gondii infection, gene regulation, apoptosis and cancer process alterations in infected spleen., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Toxoplasma gondii is a eukaryotic parasite of the phylum Apicomplexa, which infects all warm-blood animals, including humans. In the present study, we examined sequence variation in dense granule 20 (GRA20) genes among T. gondii isolates collected from different hosts and geographical regions worldwide. The complete GRA20 genes were amplified from 16 T. gondii isolates using PCR, sequence were analyzed, and phylogenetic reconstruction was analyzed by maximum parsimony (MP) and maximum likelihood (ML) methods. The results showed that the complete GRA20 gene sequence was 1,586 bp in length among all the isolates used in this study, and the sequence variations in nucleotides were 0-7.9% among all strains. However, removing the type III strains (CTG, VEG), the sequence variations became very low, only 0-0.7%. These results indicated that the GRA20 sequence in type III was more divergence. Phylogenetic analysis of GRA20 sequences using MP and ML methods can differentiate 2 major clonal lineage types (type I and type III) into their respective clusters, indicating the GRA20 gene may represent a novel genetic marker for intraspecific phylogenetic analyses of T. gondii.

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.

Toxoplasmosis, one of the most important health-threatening diseases worldwide, is caused by Toxoplasma gondii , which infects a wide range of warm-blooded animals and humans, leading to enormous health and socioeconomic concerns. T. gondii can establish chronic infection to evade the immune response in hosts. Once a chronic infection has been established, the available treatments cannot efficiently control this stage of T. gondii efficiently. Moreover, the available treatments rely only on a few drugs, such as sulfapyridine and pyrimethamine, that tend to have severe side effects. Given these factors, vaccination has been considered to be the most efficient method to prevent and control this disease. However, there is currently lack of effective vaccine available for use to prevent toxoplasmosis apart form Toxovax®, the only available vaccine, which is used in sheep to prevent abortion. To address this problem, we knocked out the NPT1 gene of the type I T. gondii strain using the CRISPR-Cas9 system, constructed a live-attenuated vaccine and evaluated its protective efficacy in a mouse model. Immunization of mice with RH:Δ NPT1 induced a high level of Toxoplasma -specific IgG1, IgG2a and total IgG 42 days after immunization. There was a significant increase in the levels of cytokines in the splenocyte suspensions of RH:Δ NPT1 -infected mice, and a mixed Th1/Th2 response was induced in the mice. Remarkably, after heterologous challenges with tachyzoites of the RH, PYS and Pru strains and cysts of the Pru strain by different infection routes, the immunized animals were protected from toxoplasmosis with a 100% survival rate, in both acute and chronic infection. In addition, compared with control mice, the Pru cyst load was clearly reduced in the brains of RH:Δ NPT1 -infected immunization-mice. Our study demonstrated that the RH:Δ NPT1 strain was able to evoke strong anti- Toxoplasma immune responses and provide effective protection against parasite strains with different levels of virulence, suggesting that the RH:Δ NPT1 strain may represent a promising live-attenuated vaccine against toxoplasmosis, which is worthy of further evaluation in food-producing animals and in definitive feline host. [ABSTRACT FROM AUTHOR]

Toxoplasma gondii can infect all warm-blooded animals including human beings. T. gondii dense granule protein 16 (TgGRA16) as a crucial virulence factor could modulate the host gene expression. Here, a DNA vaccine expressing TgGRA16 was constructed to explore the protective efficacy against T. gondii infection in Kunming mice. The immune responses induced by pVAX-GRA16 were also evaluated. Mice immunized with pVAX-GRA16 could elicit higher levels of specific IgG antibody and strong cellular response compared to those in controls. The DNA vaccination significantly increased the levels of cytokines (IFN-γ, IL-2, IL-4, and IL-10) and the percentages of CD4+ and CD8+ T cells in mice. After lethal challenge, mice immunized with pVAX-GRA16 (8.4±0.78 days) did not show a significant longer survival time than that in controls (7.1±0.30 days) (p>0.05). However, in chronic toxoplasmosis model (administration of 10 brain cysts of PRU strain orally), numbers of tissue cysts in mice immunized with pVAX-GRA16 were significantly reduced compared to those in controls (p<0.05) and the rate of reduction could reach 43.89%. The results indicated that the TgGRA16 would be a promising vaccine candidate for further development of effective epitope-based vaccines against chronic T. gondii infection in mice. [ABSTRACT FROM AUTHOR]

Toxoplasma gondii , an obligatory intracellular protozoan, can cause serious public health problems and economic losses worldwide. Two novel dense granule proteins (GRA17 and GRA23) were recently identified as T. gondii -secreted proteins which are localized to the parasitophorous vacuole membrane (PVM) and can mediate the movement of small molecules between the host cell and parasitophorous vacuole (PV). In the present study, we evaluated the protective immunity induced by DNA vaccination with genes encoding GRA17 and GRA23 against acute toxoplasmosis in mice. Eukaryotic expressing plasmids pVAX-TgGRA17 and pVAX-TgGRA23 were constructed. Then, BALB/c mice were intramuscularly immunized with pVAX-TgGRA17, pVAX-TgGRA23, or pVAX-TgGRA17 + pVAX-TgGRA23 followed by challenge infection with the highly virulent RH strain of T. gondii . The specific immune responses and protective efficacy against T. gondii were examined by cytokine and serum antibody measurements, lymphocyte proliferation assays, flow cytometry of lymphocytes and the survival time after challenge. Our results showed that mice immunized with pVAX-TgGRA17, pVAX-TgGRA23, or pVAX-TgGRA17 + pVAX-TgGRA23 induced specific humoral and cellular responses, with higher level of IgG antibody, increased levels of Th1-type cytokines IFN-γ and IL-12 (p70), and CD3 + CD4 + CD8 − and CD3 + CD8 + CD4 − T cells, as well as prolonged survival time (9.1 ± 0.32 days for pVAX-TgGRA17, 10.8 ± 0.79 days for pVAX-TgGRA23, and 12.6 ± 2.55 days for pVAX-TgGRA17 + pVAX-TgGRA23) compared to the blank control (7.11 ± 0.33 days), PBS control (7.22 ± 0.44 days), and pVAX I control (7.11 ± 0.33 days). These results demonstrated that both TgGRA17 and TgGRA23 are potential vaccine candidates, TgGRA23 has a better immunogenicity, and co-immunization of pVAX-TgGRA17 and pVAX-TgGRA23 induces better protective efficacy. [ABSTRACT FROM AUTHOR]

Toxoplasma gondii , an obligate intracellular parasite, is able to infect many animal species and humans, and can cause toxoplasmosis of the host. In this study, we examined sequence variation in rhoptry protein 47 (ROP47) gene among T. gondii isolates originating from different hosts and geographical regions. The entire genome region of the ROP47 gene was amplified and sequenced, and phylogenetic relationship was reconstructed using maximum parsimony (MP), maximum likelihood (ML) and neighbor-joining (NJ), based on the ROP47 gene sequences. The results of sequence alignments showed that all ROP47 gene sequences were 396 bp in length. There were 19 variable nucleotide positions in the coding region, resulted in 16 amino acid substitutions (12.21%) among all examined T. gondii strains and the existence of polymorphic restriction sites for endonucleases SacI and AflIII, allowing the differentiation of the three major clonal lineage types I, II and III by PCR-RFLP. Phylogenetic analysis of ROP47 gene sequences showed that three major clonal lineage types I, II and III were clustered differently, consistent with PCR-RFLP results. These results suggest that ROP47 gene sequence may represent a potential novel genetic marker for population genetic studies of T. gondii isolates. [ABSTRACT FROM AUTHOR]

In the present study, a dense granule protein 17 (gra17) and novel putative transporter (npt1) double deletion mutant of Toxoplasma gondii RH strain was engineered. The protective efficacy of vaccination using RHΔgra17Δnpt1 tachyzoites against acute, chronic, and congenital toxoplasmosis was studied in a mouse model. Immunization using RHΔgra17Δnpt1 induced a strong humoral and cellular response, as indicated by the increased levels of anti-T. gondii specific IgG, interleukin 2 (IL-2), IL-10, IL-12, and interferon-gamma (IFN-γ). Vaccinated mice were protected against a lethal challenge dose (103 tachyzoites) of wild-type homologous (RH) strain and heterologous (PYS and TgC7) strains, as well as against 100 tissue cysts or oocysts of Pru strain. Vaccination also conferred protection against chronic infection with 10 tissue cysts or oocysts of Pru strain, where the numbers of brain cysts in the vaccinated mice were significantly reduced compared to those detected in the control (unvaccinated + infected) mice. In addition, vaccination protected against congenital infection with 10 T. gondii Pru oocysts (administered orally on day 5 of gestation) as shown by the increased litter size, survival rate and the bodyweight of pups born to vaccinated dams compared to those born to unvaccinated + infected dams. The brain cyst burden of vaccinated dams was significantly lower than that of unvaccinated dams infected with oocysts. Our data show that T. gondii RHΔgra17Δnpt1 mutant strain can protect mice against acute, chronic, and congenital toxoplasmosis by balancing inflammatory response with immunogenicity. [ABSTRACT FROM AUTHOR]

Toxoplasma gondii is an intracellular zoonotic parasite that causes toxoplasmosis, which can cause economic losses and serious public health problems worldwide. A member of the T. gondii calcium-dependent protein kinases family, TgCDPK1 was recently identified as an essential regulator of exocytosis in T. gondii, and participated in direct parasite motility, host-cell invasion and egress. In the present study, the protective immunity of recombinant TgCDPK1 protein (rTgCDPK1) was evaluated against acute toxoplasmosis in mice. rTgCDPK1 were expressed and purified, BABL/c mice were intraperitoneally immunized with rTgCDPK1 and challenged with the highly virulent RH strain of T. gondii. The specific immune responses were analyzed by measuring the cytokine and serum antibody, and lymphocyte proliferation assays, flow cytometry of lymphocytes and the survival curve were employed to evaluate the protective efficacy. From the results we found that special humoral and cellular responses could be elicited in vaccine mice, and higher level of IgG antibody, and the significant increased levels of Th1-type cytokines IFN-γ, IL-12 (p70), IL10 and CD3+CD4+CD8− and CD3+CD8+CD4− T cells could also be detected comparing to control mice (P < 0.05). All vaccinated mice prolonged survival time (14.90 ± 2.89 days) challenge with 1000 tachyzoites of RH, while the control mice died within 8 days. These results indicated that TgCDPK1 protein was a potential vaccine candidate against acute toxoplasmosis. • The rTgCDPK1 could elicited special humoral and cellular responses in vaccine mice. • All vaccinated mice prolonged survival time challenge with tachyzoites of RH. • TgCDPK1 protein is a potential vaccine candidate against acute toxoplasmosis. [ABSTRACT FROM AUTHOR]