OBJECTIVE: To analyze the RNA binding protein of Toxoplasma gondii (TgDDX39) using bioinformatics technology, and to evaluate the immunogenicity of TgDDX39, so as to provide insights into development of toxoplasmosis vaccines. METHODS: The amino acid sequences of TgDDX39 were retrieved from the ToxoDB database, and the physicochemical properties, transmembrane structure domain, signal peptide sites, post-translational modification sites, coils, secondary and tertiary structures, hydrophobicity, and antigenic epitopes of the TgDDX39 protein were predicted using online bioinformatics tools, incluiding ProtParam, TMHMM 2.0, SignalP 5.0, NetPhos 3.1, COILS, SOPMA, Phyre2, ProtScale, ABCpred, SYFPEITHI and DNA-STAR. RESULTS: TgDDX39 protein was predicted to be an unstable hydrophilic protein with the molecular formula of C₂₁₇₃H₃₄₅₈N₅₉₈O₆₆₁S₁₈, which contained 434 amino acids and had an estimated molecular weight of 49.1 kDa and a theoretical isoelectric point of 5.55. The protein was predicted to have an extremely low possibility of signal peptides, without transmembrane regions, and contain 27 phosphorylation sites. The β turn and random coils accounted for 39.63% of the secondary structure of the TgDDX39 protein, and a coiled helix tended to produce in one site. In addition, the TgDDX39 protein contained multiple B and T cell antigenic epitopes. CONCLUSIONS Bioinformatics analyses predict that TgDDX39 protein has high immunogenicity and contains multiple antigenic epitopes. TgDDX39 protein is a potential candidate antigen for vaccine development.
Humans ; Toxoplasma/metabolism* ; Toxoplasmosis/prevention & control* ; Vaccines ; Epitopes, T-Lymphocyte ; Computational Biology ; Protozoan Proteins/chemistry*

Specific gene expressions of host cells by spontaneous STAT6 phosphorylation are major strategy for the survival of intracellular Toxoplasma gondii against parasiticidal events through STAT1 phosphorylation by infection provoked IFN-γ. We determined the effects of small molecules of tyrosine kinase inhibitors (TKIs) on the growth of T. gondii and on the relationship with STAT1 and STAT6 phosphorylation in ARPE-19 cells. We counted the number of T. gondii RH tachyzoites per parasitophorous vacuolar membrane (PVM) after treatment with TKIs at 12-hr intervals for 72 hr. The change of STAT6 phosphorylation was assessed via western blot and immunofluorescence assay. Among the tested TKIs, Afatinib (pan ErbB/EGFR inhibitor, 5 µM) inhibited 98.0% of the growth of T. gondii, which was comparable to pyrimethamine (5 µM) at 96.9% and followed by Erlotinib (ErbB1/EGFR inhibitor, 20 µM) at 33.8% and Sunitinib (PDGFR or c-Kit inhibitor, 10 µM) at 21.3%. In the early stage of the infection (2, 4, and 8 hr after T. gondii challenge), Afatinib inhibited the phosphorylation of STAT6 in western blot and immunofluorescence assay. Both JAK1 and JAK3, the upper hierarchical kinases of cytokine signaling, were strongly phosphorylated at 2 hr and then disappeared entirely after 4 hr. Some TKIs, especially the EGFR inhibitors, might play an important role in the inhibition of intracellular replication of T. gondii through the inhibition of the direct phosphorylation of STAT6 by T. gondii.
Antiparasitic Agents/pharmacology ; Blotting, Western ; Cell Line ; Enzyme Activation/drug effects ; Fluorescent Antibody Technique ; Humans ; Janus Kinase 1/metabolism ; Janus Kinase 3/metabolism ; Phosphorylation/drug effects ; Quinazolines/*pharmacology ; STAT6 Transcription Factor/*metabolism ; Signal Transduction/*drug effects ; Toxoplasma/*drug effects/physiology ; Toxoplasmosis/physiopathology

The sodium hydrogen exchanger 1 (NHE1), which functions in maintaining the ratio of Na+ and H+ ions, is widely distributed in cell plasma membranes. It plays a prominent role in pH balancing, cell proliferation, differentiation, adhesion, and migration. However, its exact subcellular location and biological functions in Toxoplasma gondii are largely unclear. In this study, we cloned the C-terminal sequence of T. gondii NHE1 (TgNHE1) incorporating the C-terminal peptide of NHE1 (C-NHE1) into the pGEX4T-1 expression plasmid. The peptide sequence was predicted to have good antigenicity based on the information obtained from an immune epitope database. After induction of heterologous gene expression with isopropyl-b-D-thiogalactoside, the recombinant C-NHE1 protein successfully expressed in a soluble form was purified by glutathione sepharose beads as an immunogen for production of a rabbit polyclonal antiserum. The specificity of this antiserum was confirmed by western blotting and immunofluorescence. The antiserum could reduce T. gondii invasion into host cells, indicated by the decreased TgNHE1 expression in T. gondii parasites that were pre-incubated with antiserum in the process of cell entry. Furthermore, the antiserum reduced the virulence of T. gondii parasites to host cells in vitro, possibly by blocking the release of Ca2+. In this regard, this antiserum has potential to be a valuable tool for further studies of TgNHE1.
Animals ; Cell Line ; Immune Sera/genetics/immunology/*metabolism ; Male ; Mice ; Protozoan Proteins/genetics/*metabolism ; Rabbits ; Recombinant Proteins/immunology ; Sheep ; Sodium-Hydrogen Antiporter/genetics/immunology/*metabolism ; Toxoplasma/genetics/immunology/*metabolism ; Toxoplasmosis/parasitology/prevention & control

Toxoplasmosis is a serious disease caused by Toxoplasma gondii, one of the most widespread parasites in the world. Lipid metabolism is important in the intracellular stage of T. gondii. Stearoyl-CoA desaturase (SCD), a key enzyme for the synthesis of unsaturated fatty acid is predicted to exist in T. gondii. Sterculic acid has been shown to specifically inhibit SCD activity. Here, we examined whether sterculic acid and its methyl ester analogues exhibit anti-T. gondii effects in vitro. T. gondii-infected Vero cells were disintegrated at 36 hr because of the propagation and egress of intracellular tachyzoites. All test compounds inhibited tachyzoite propagation and egress, reducing the number of ruptured Vero cells by the parasites. Sterculic acid and the methyl esters also inhibited replication of intracellular tachyzoites in HFF cells. Among the test compounds, sterculic acid showed the most potent activity against T. gondii, with an EC50 value of 36.2 μM, compared with EC50 values of 248-428 μM for the methyl esters. Our study demonstrated that sterculic acid and its analogues are effective in inhibition of T. gondii growth in vitro, suggesting that these compounds or analogues targeting SCD could be effective agents for the treatment of toxoplasmosis.
Esters ; Lipid Metabolism ; Parasites ; Stearoyl-CoA Desaturase ; Toxoplasma* ; Toxoplasmosis ; Vero Cells

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.
Animals ; Base Sequence ; Brazil ; China ; Deer ; *Genetic Variation ; Genotype ; Goats ; Humans ; Molecular Sequence Data ; Phylogeny ; Protozoan Proteins/*genetics/metabolism ; Sheep ; Swine ; Toxoplasma/classification/*genetics/isolation & purification/parasitology/physiology ; Toxoplasmosis/*parasitology ; Toxoplasmosis, Animal/*parasitology ; United States

Toxoplasma gondii is an opportunistic protozoan parasite that can infect almost all warm-blooded animals including humans with a worldwide distribution. Micronemes play an important role in invasion process of T. gondii, associated with the attachment, motility, and host cell recognition. In this research, sequence diversity in microneme protein 6 (MIC6) gene among 16 T. gondii isolates from different hosts and geographical regions and 1 reference strain was examined. The results showed that the sequence of all the examined T. gondii strains was 1,050 bp in length, and their A + T content was between 45.7% and 46.1%. Sequence analysis presented 33 nucleotide mutation positions (0-1.1%), resulting in 23 amino acid substitutions (0-2.3%) aligned with T. gondii RH strain. Moreover, T. gondii strains representing the 3 classical genotypes (Type I, II, and III) were separated into different clusters based on the locus of MIC6 using phylogenetic analyses by Bayesian inference (BI), maximum parsimony (MP), and maximum likelihood (ML), but T. gondii strains belonging to ToxoDB #9 were separated into different clusters. Our results suggested that MIC6 gene is not a suitable marker for T. gondii population genetic studies.
Amino Acid Sequence ; Animals ; Base Sequence ; Cats ; Cell Adhesion Molecules/chemistry/*genetics/metabolism ; Deer ; *Genetic Variation ; Genotype ; Goats ; Humans ; Molecular Sequence Data ; Phylogeny ; Protozoan Proteins/chemistry/*genetics/metabolism ; Sequence Alignment ; Sheep ; Swine ; Toxoplasma/classification/*genetics/isolation & purification/physiology ; Toxoplasmosis/*parasitology ; Toxoplasmosis, Animal/*parasitology

Toxoplasma gondii, an obligate intracellular protozoan parasite of the phylum Apicomplexa, can infect all warm-blooded vertebrates, including humans, livestock, and marine mammals. The aim of this study was to investigate whether superoxide dismutase (SOD) of T. gondii can be used as a new marker for genetic study or a potential vaccine candidate. The partial genome region of the SOD gene was amplified and sequenced from 10 different T. gondii isolates from different parts of the world, and all the sequences were examined by PCR-RFLP, sequence analysis, and phylogenetic reconstruction. The results showed that partial SOD gene sequences ranged from 1,702 bp to 1,712 bp and A + T contents varied from 50.1% to 51.1% among all examined isolates. Sequence alignment analysis identified total 43 variable nucleotide positions, and these results showed that 97.5% sequence similarity of SOD gene among all examined isolates. Phylogenetic analysis revealed that these SOD sequences were not an effective molecular marker for differential identification of T. gondii strains. The research demonstrated existence of low sequence variation in the SOD gene among T. gondii strains of different genotypes from different hosts and geographical regions.
Amino Acid Sequence ; Animals ; Base Sequence ; Cats ; *Genetic Variation ; Goats ; Humans ; Molecular Sequence Data ; Phylogeny ; Protozoan Proteins/chemistry/*genetics/metabolism ; Sequence Alignment ; Sheep ; Superoxide Dismutase/chemistry/*genetics/metabolism ; Toxoplasma/classification/*enzymology/genetics/isolation & purification ; Toxoplasmosis/*parasitology ; Toxoplasmosis, Animal/*parasitology

BACKGROUNDAlthough the onset of anemia during infectious disease is commonly correlated with production of inflammatory cytokines, the mechanisms by which cytokines induce anemia are poorly defined. This study focused on the mechanism research.

METHODSDifferent types of mice were infected perorally with Toxoplasma gondii strain ME49. At the indicated times, samples from each mouse were harvested, processed, and analyzed individually. Blood samples were analyzed using a Coulter Counter and red blood cell (RBC) survival was measured by biotinylation. Levels of tumor necrosis factor-α (TNF-α), inducible nitric oxide synthase (iNOS), and inducible protein 10 (IP-10) mRNA in liver tissue were measured by real-time polymerase chain reaction.

RESULTST. gondii-infected mice exhibited anemia due to a decrease in both erythropoiesis and survival time of RBC in the circulation (P < 0.02). In addition, infection-stimulated anemia was associated with fecal occult, supporting previous literature that hemorrhage is a consequence of T. gondii infection in mice. Infection-induced anemia was abolished in interferon gamma (IFNγ) and IFNγ receptor deficient mice (P < 0.05) but was still evident in mice lacking TNF-α, iNOS, phagocyte NADPH oxidase or IP-10 (P < 0.02). Neither signal transducer and activator of transcription 1 (STAT1) deficient mice nor 129S6 controls exhibited decreased erythropoiesis, but rather suffered from an anemia resulting solely from increased loss of circulating RBC.

CONCLUSIONSInfection-stimulated decrease in erythropoiesis and losses of RBC have distinct mechanistic bases. These results show that during T. gondii infection, IFNγ is responsible for an anemia that results from both a decrease in erythropoiesis and a STAT1 independent loss of circulating RBC.

Anemia ; genetics ; metabolism ; Animals ; Erythrocytes ; pathology ; Interferon-gamma ; metabolism ; Male ; Mice ; Mice, Knockout ; Nitric Oxide Synthase Type II ; genetics ; metabolism ; Receptors, Interferon ; genetics ; metabolism ; STAT1 Transcription Factor ; genetics ; metabolism ; Toxoplasma ; pathogenicity ; Tumor Necrosis Factor-alpha ; genetics ; metabolism

Recombinant antigenic proteins of Toxoplasma gondii are alternative source of antigens which are easily obtainable for serodiagnosis of toxoplasmosis. In this study, highly antigenic secretory organellar proteins, dense granular GRA2 and GRA3, rhoptrial ROP2, and micronemal MIC2, were analyzed by bioinformatics approach to express as water-soluble forms of antigenic domains. The transmembrane region and disorder tendency of 4 secretory proteins were predicted to clone the genes into pGEX-4T-1 vector. Recombinant plasmids were transformed into BL21 (DE3) pLysS E. coli, and GST fusion proteins were expressed with IPTG. As a result, GST fusion proteins with GRA225-105, GRA339-138, ROP2324-561, and MIC21-284 domains had respectively higher value of IgG avidity. The rGST-GRA225-105 and rGST-GRA339-138 were soluble, while rGST-ROP2324-561 and rGST-MIC21-284 were not. GRA231-71, intrinsically unstructured domain (IUD) of GRA2, was used as a linker to enhance the solubility. The rGST-GRA231-71-ROP2324-561, a chimeric protein, appeared to be soluble. Moreover, rGST-GRA231-71-MIC21-284 was also soluble and had higher IgG avidity comparing to rGST-MIC21-284. These 4 highly expressed and water-soluble recombinant antigenic proteins may be promising candidates to improve the serodiagnosis of toxoplasmosis in addition to the major surface antigen of SAG1.
Animals ; Antibodies, Protozoan/immunology ; Antibody Affinity ; Antigens, Protozoan/chemistry/*diagnostic use/genetics/immunology ; *Gene Expression ; Immunoglobulin G/blood/immunology ; Mice, Inbred BALB C ; Recombinant Proteins/chemistry/*diagnostic use/genetics/immunology ; Serologic Tests/methods ; Solubility ; Toxoplasma/genetics/immunology/*metabolism ; Toxoplasmosis/diagnosis

OBJECTIVEThe protozoan Toxoplasma gondii expresses large amounts of a 37 kDa Type 2C serine-threonine phosphatase, the so-called TgPP2C which has been suggested to contribute to parasite growth regulation. Ectopic expression in mammalian cells also indicated that the enzyme could regulate growth and survival. In this study, we aimed to investigate the interaction of TgPP2C with human SSRP1 (structure-specific recognition protein 1) and the effects of TgPP2C on cell viability.

METHODSThe yeast two hybrid system, His-tag pull-down and co-immunoprecipitation assays were used to confirm the interaction of TgPP2C with SSRP1 and determine the binding domain on SSRP1. The evaluation of cell apoptosis was performed using cleaved caspase-3 antibody and Annexin-V/PI kit combined with flow cytometry.

RESULTSWe identified human SSRP1 as an interacting partner of TgPP2C. The C-terminal region of SSRP1 including the amino acids 471 to 538 was specifically mapped as the region responsible for interaction with TgPP2C. The overexpression of TgPP2C down-regulated cell apoptosis and negatively regulated apoptosis induced by DRB, casein kinase II (CKII) inhibitor, through enhanced interaction with SSRP1.

CONCLUSIONTgPP2C may be a parasitic factor capable of promoting cell survival through interaction with the host protein SSRP1, thereby creating a favorable environment for parasite growth.

Apoptosis ; Blotting, Western ; DNA-Binding Proteins ; genetics ; metabolism ; Flow Cytometry ; HeLa Cells ; High Mobility Group Proteins ; genetics ; metabolism ; Humans ; Immunoprecipitation ; Phosphoprotein Phosphatases ; genetics ; metabolism ; Protein Phosphatase 2C ; Toxoplasma ; enzymology ; Transcriptional Elongation Factors ; genetics ; metabolism ; Two-Hybrid System Techniques