We studied on the proteomic characteristics of Toxoplasma gondii KI-1 tachyzoites which were originally isolated from a Korean patient, and compared with those of the well-known virulent RH strain using 2-dimensional electrophoresis (2-DE), mass spectrometry, and quantitative real-time PCR. Two-dimensional separation of the total proteins isolated from KI-1 tachyzoites revealed up to 150 spots, of which 121 were consistent with those of RH tachyzoites. Of the remaining 29 spots, 14 showed greater than 5-fold difference in density between the KI-1 and RH tachyzoites at a pH of 5.0-8.0. Among the 14 spots, 5 from the KI-1 isolate and 7 from the RH strain were identified using MALDI-TOF mass spectrometry and database searches. The spots from the KI-1 tachyzoites were dense granule proteins (GRA 2, 3, 6, and 7), hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGRPTase), and uracil phosphoribosyltransferase (UPRTase). The spots from the RH strain were surface antigen 1 (SAG 1), L-lactate dehydrogenase (LDH), actin, chorismate synthase, peroximal catalase, hexokinase, bifunctional dihydrofolate reductase-thymidylate synthase (DHTR-TS), and nucleoside-triphosphatases (NTPases). Quantitative real-time PCR supported our mass spectrometric results by showing the elevated expression of the genes encoding GRA 2, 3, and 6 and UPRTase in the KI-1 tachyzoites and those encoding GRA 7, SAG 1, NTPase, and chorismate synthase in the RH tachyzoites. These observations demonstrate that the protein compositions of KI-1 and RH tachyzoites are similar but differential protein expression is involved in virulence.
Electrophoresis, Gel, Two-Dimensional ; Gene Expression Regulation, Developmental ; Humans ; Molecular Sequence Data ; *Proteomics ; Protozoan Proteins/chemistry/*genetics/metabolism ; Toxoplasma/chemistry/*genetics/*growth & development/metabolism ; Toxoplasmosis/parasitology

OBJECTIVETo establish an tachyzoite-brachyzoite interconversion system for Toxoplasma gondii RH strain in vitro.

METHODSCOS-7 cells were inoculated with purified tachyzoites of T.gondii RH strain and cultured in vitro. The morphology of the cultured cells and parasites was observed and the total cellular RNA extracted on days 1 to 6 following the inoculation for detecting the expression of tachyzoite-specific protein (SAG1) and bradyzoite-specific proteins (BAG1 and SAG2C) using RT-PCR.

RESULTSWith the passage of time, the number of parasites in COS-7 cells increased but the proliferation rate was lowered gradually. The intracellular tachyzoites proliferated by means of budding and binary fission, which led to the changes in the alignment of the parasites in the cells from curved pairs, rosette or clustered, and semi-circular patterns to spherical encapsulation-like structures. These changes indicated the gradual transformation of the tachyzoites into bradyzoites. The expressions of the tachyzoite-specific SAG1 gene were detected throughout the 6 days of in vitro culture. The expression of the bradyzoite-specific BAG1 gene had been detected since the second day after the inoculation and SAG2C gene since the fifth day. Alteration of the culture condition resulted in gradual transformation of the bradyzoites into tachyzoites.

CONCLUSIONAn in vitro tachzoites-bradyzoite interconversion system for T.gondii has been successfully established, which provides the basis for further study of the mechanism of interconversion.

Animals ; COS Cells ; Cell Culture Techniques ; Cercopithecus aethiops ; Cysts ; Female ; Genes, Protozoan ; genetics ; Host-Parasite Interactions ; Mice ; Protozoan Proteins ; biosynthesis ; genetics ; Toxoplasma ; growth & development ; physiology

Toxoplasma gondii is a protozoan parasite with a broad range of intermediate hosts. Chickens as important food-producing animals can also serve as intermediate hosts. To date, experimental studies on the pathogenicity of T. gondii in broiler chickens were rarely reported. The objective of the present study was to compare the pathogenicity of 5 different T. gondii strains (RH, CN, JS, CAT2, and CAT3) from various host species origin in 10-day-old chickens. Each group of chickens was infected intraperitoneally with 5 x 10(8), 1 x 10(8), 1 x 10(7), and 1 x 10(6) tachyzoites of the 5 strains, respectively. The negative control group was mockly inoculated with PBS alone. After infection, clinical symptoms and rectal temperatures of all the chickens were checked daily. Dead chickens during acute phage of the infection were checked for T. gondii tachyzoites by microscope, while living cases were checked for T. gondii infection at day 53 post-inoculation (PI) by PCR method. Histopathological sections were used to observe the pathological changes in the dead chickens and the living animals at day 53 PI. No significant differences were found in survival periods, histopathological findings, and clinical symptoms among the chickens infected with the RH, CN, CAT2, and CAT3 strains. Histopathological findings and clinical symptoms of the JS (chicken origin) group were similar to the others. However, average survival times of infected chickens of the JS group inoculated with 5 x 10(8) and 1 x 10(8) tachyzoites were 30.0 and 188.4 hr, respectively, significantly shorter than those of the other 4 mammalian isolates. Chickens exposed to 10(8) of T. gondii tachyzoites and higher showed acute signs of toxoplasmosis, and the lesions were relatively more severe than those exposed to lower doses. The results indicated that the pathogenicity of JS strain was comparatively stronger to the chicken, and the pathogenicity was dose-dependent.
Animals ; Antibodies, Protozoan/blood ; Cat Diseases/parasitology ; Cats ; Chickens ; Poultry Diseases/blood/mortality/*parasitology/pathology ; Swine ; Swine Diseases/parasitology ; Toxoplasma/genetics/growth & development/*pathogenicity/physiology ; Toxoplasmosis, Animal/blood/mortality/*parasitology/pathology ; Virulence

Toxoplasma gondii can modulate host cell gene expression; however, determining gene expression levels in intermediate hosts after T. gondii infection is not known much. We selected 5 genes (ALDH1A2, BEX2, CCL3, EGR2 and PLAU) and compared the mRNA expression levels in the spleen, liver, lung and small intestine of genetically different mice infected with T. gondii. ALDH1A2 mRNA expressions of both mouse strains were markedly increased at day 1-4 postinfection (PI) and then decreased, and its expressions in the spleen and lung were significantly higher in C57BL/6 mice than those of BALB/c mice. BEX2 and CCR3 mRNA expressions of both mouse strains were significantly increased from day 7 PI and peaked at day 15-30 PI (P<0.05), especially high in the spleen liver or small intestine of C57BL/6 mice. EGR2 and PLAU mRNA expressions of both mouse strains were significantly increased after infection, especially high in the spleen and liver. However, their expression patterns were varied depending on the tissue and mouse strain. Taken together, T. gondii-susceptible C57BL/6 mice expressed higher levels of these 5 genes than did T. gondii-resistant BALB/c mice, particularly in the spleen and liver. And ALDH1A2 and PLAU expressions were increased acutely, whereas BEX2, CCL3 and EGR2 expressions were increased lately. Thus, these demonstrate that host genetic factors exert a strong impact on the expression of these 5 genes and their expression patterns were varied depending on the gene or tissue.
Aldehyde Dehydrogenase/*genetics/metabolism ; Animals ; Brain/metabolism/parasitology ; Chemokine CCL3/*genetics/metabolism ; Early Growth Response Protein 2/*genetics/metabolism ; Gene Expression Profiling ; Humans ; Lung/metabolism/parasitology ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Mice, Inbred Strains ; Nerve Tissue Proteins/*genetics/metabolism ; Organ Specificity ; Spleen/metabolism/virology ; Toxoplasma/*physiology ; Toxoplasmosis/*genetics/metabolism/parasitology ; Urokinase-Type Plasminogen Activator/*genetics/metabolism

PURPOSE: Surface antigen 3 (SAG3) of Toxoplasma gondii is very similar in structure to the major surface antigen 1 (SAG1). Although numerous studies have supported the importance of SAG1 in protection against T. gondii infection, few reports exist on SAG3. MATERIALS AND METHODS: Glutathione-S-transferase (GST)-fused SAG3 of T. gondii (rSAG3) were immunized into BALB/c mice alone or in combination with Quil A (rSAG3/Quil A), and then evaluated the protective immunity in vivo and in vitro against murine toxoplasmosis. RESULTS: Immunization with rSAG3 or rSAG3/Quil A resulted in significantly more survival days and fewer brain cysts after challenge with T. gondii compared to an infected control group. Mice immunized with rSAG3 alone or in combination with Quil A produced significantly more specific IgG2a antibody, whereas specific IgG1 antibody titers did not increase. The percentage of CD8+ T cells, IFN-gamma mRNA expression, and nitric oxide production significantly increased in rSAG3- and rSAG3/Quil A-immunized mice. CONCLUSION: These results indicate that vaccination with Toxoplasma rSAG3 results in partial protective immunity against T. gondii infection through induction of a Th1-type immune response, and that protective immunity is accelerated by the modulating effects of Quil A.
Animals ; Antigens, Protozoan/genetics/*immunology/metabolism ; Bacterial Proteins/genetics/immunology/metabolism ; Blotting, Western ; Enzyme-Linked Immunosorbent Assay ; Female ; Flow Cytometry ; Immunoglobulin G/immunology ; Interferon-gamma/metabolism ; Mice ; Mice, Inbred BALB C ; Nitric Oxide/metabolism ; Protozoan Proteins/genetics/immunology/metabolism ; Recombinant Fusion Proteins/genetics/immunology/metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Saponins/*immunology ; Toxoplasma/growth & development/*immunology ; Toxoplasmosis, Animal/*immunology/metabolism/microbiology ; Vaccination/*methods

PURPOSE: Surface antigen 3 (SAG3) of Toxoplasma gondii is very similar in structure to the major surface antigen 1 (SAG1). Although numerous studies have supported the importance of SAG1 in protection against T. gondii infection, few reports exist on SAG3. MATERIALS AND METHODS: Glutathione-S-transferase (GST)-fused SAG3 of T. gondii (rSAG3) were immunized into BALB/c mice alone or in combination with Quil A (rSAG3/Quil A), and then evaluated the protective immunity in vivo and in vitro against murine toxoplasmosis. RESULTS: Immunization with rSAG3 or rSAG3/Quil A resulted in significantly more survival days and fewer brain cysts after challenge with T. gondii compared to an infected control group. Mice immunized with rSAG3 alone or in combination with Quil A produced significantly more specific IgG2a antibody, whereas specific IgG1 antibody titers did not increase. The percentage of CD8+ T cells, IFN-gamma mRNA expression, and nitric oxide production significantly increased in rSAG3- and rSAG3/Quil A-immunized mice. CONCLUSION: These results indicate that vaccination with Toxoplasma rSAG3 results in partial protective immunity against T. gondii infection through induction of a Th1-type immune response, and that protective immunity is accelerated by the modulating effects of Quil A.
Animals ; Antigens, Protozoan/genetics/*immunology/metabolism ; Bacterial Proteins/genetics/immunology/metabolism ; Blotting, Western ; Enzyme-Linked Immunosorbent Assay ; Female ; Flow Cytometry ; Immunoglobulin G/immunology ; Interferon-gamma/metabolism ; Mice ; Mice, Inbred BALB C ; Nitric Oxide/metabolism ; Protozoan Proteins/genetics/immunology/metabolism ; Recombinant Fusion Proteins/genetics/immunology/metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Saponins/*immunology ; Toxoplasma/growth & development/*immunology ; Toxoplasmosis, Animal/*immunology/metabolism/microbiology ; Vaccination/*methods