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Infection with the apicomplexan protozoan parasite Toxoplasma gondii is an ongoing public health problem. The parasite's ability to invade and replicate within the host cell is dependent on many effectors, such as dense granule proteins (GRAs) released from the specialized organelle dense granules, into host cells. GRAs have emerged as important determinants of T. gondii pathogenesis. However, the functions of some GRAs remain undefined. In this study, we used CRISPR-Cas9 technique to disrupt 17 GRA genes (GRA11, GRA12 bis, GRA13, GRA14, GRA20, GRA21, GRA28-31, GRA33-38 , and GRA40) in the virulent T. gondii RH strain. The CRISPR-Cas9 constructs abolished the expression of the 17 GRA genes. Functional characterization of single Δ GRA mutants was achieved in vitro using cell-based plaque assay and egress assay, and in vivo in BALB/c mice. Targeted deletion of these 17 GRA genes had no significant effect neither on the in vitro growth and egress of the mutant strains from the host cells nor on the parasite virulence in the mouse model of infection. Comparative analysis of the transcriptomics data of the 17 GRA genes suggest that GRAs may serve different functions in different genotypes and life cycle stages of the parasite. In sum, although these 17 GRAs might not be essential for RH strain growth in vitro or virulence in mice, they may have roles in other strains or parasite stages, which warrants further investigations. [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 is a global pathogen that infects a wide range of animals and humans. During T. gondii infection, the spleen plays an important role in coordinating the adaptive and innate immune responses. However, there is little information regarding the changes in global gene expression within the spleen following T. gondii infection. To address this gap in knowledge, we examined the transcriptome of the mouse spleen following T. gondii infection. We observed differential expression of 2310 transcripts under these conditions. Analysis of KEGG and GO enrichment indicated that T. gondii alters multiple immune signaling cascades. Most of differentially expressed GO terms and pathways were downregulated, while immune-related GO terms and pathways were upregulated with response to T. gondii infection in mouse spleen. Most cytokines were upregulated in infected spleens, and all differentially expressed chemokines were upregulated which enhanced the immune cells chemotaxis to promote recruitment of immune cells, such as neutrophils, eosinophils, monocytes, dendritic cells, macrophages, NK cells, basophils, B cells, and T cells. Although IFN-γ-induced IDO (Ido1) was upregulated in the present study, it may not contribute a lot to the control of T. gondii because most differentially expressed genes involved in tryptophan metabolism pathway were downregulated. Innate immunity pathways, including cytosolic nucleic acid sensing pathway and C-type lectins-Syk-Card9 signaling pathways, were upregulated. We believe our study is the first comprehensive attempt to define the host transcriptional response to T. gondii infection in the mouse spleen. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 ± 1.15 days for pVAX-ROP54 vs 6.7 ± 0.48 days for pVAX I, 6.8 ± 0.42 days 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. [ABSTRACT FROM AUTHOR]