ABSTRACT
The DnaK-tetratricopeptide repeat (DnaK-TPR) gene (ToxoDB ID, TGME49_002020) is expressed predominantly at the bradyzoite stage. DnaK-TPR protein has a heat shock protein (DnaK) and tetratricopeptide repeat (TPR) domains with amino acid sequence similarity to the counterparts of other organisms (40.2-43.7% to DnaK domain and 41.1-66.0% to TPR domain). These findings allowed us to infer that DnaK-TPR protein is important in the tachyzoite-to-bradyzoite development or maintenance of cyst structure although the function of this gene is still unknown. An immunofluorescence assay (IFA) revealed that DnaK-TPR protein was expressed in Toxoplasma gondii-encysted and in vitro-induced bradyzoites and distributed in the whole part of parasite cells. We conducted yeast two-hybrid screening to identify proteins interacting with DnaK-TPR protein, and demonstrated that DnaK-TPR protein interacts with p23 co-chaperone protein (Tgp23). It was expected that DnaK-TPR protein would have a function as a molecular chaperon in bradyzoite cells associated with Tgp23. Possible mechanisms for this gene are discussed.
Subject(s)
Molecular Chaperones/metabolism , Protein Interaction Domains and Motifs , Protozoan Proteins/metabolism , Toxoplasma/metabolism , Amino Acid Sequence , Animals , Base Sequence , DNA Primers/chemistry , Female , HEK293 Cells , Humans , Immunoprecipitation , Male , Mice , Mice, Inbred BALB C , Mice, Inbred ICR , Mitochondria/chemistry , Molecular Chaperones/chemistry , Molecular Chaperones/genetics , Molecular Sequence Data , Oligonucleotide Probes/chemistry , Protozoan Proteins/chemistry , Protozoan Proteins/genetics , Rabbits , Toxoplasma/genetics , Toxoplasmosis, Animal/parasitologyABSTRACT
Toxoplasma gondii undergoes stage conversion from tachyzoites to bradyzoites in intermediate hosts. There have been many reports on bradyzoite-specific genes which are thought to be involved in stage conversion. Here, we described a novel T. gondii deoxyribose phosphate aldolase-like gene (TgDPA) expressing predominantly in bradyzoites. The TgDPA gene encodes 286 amino acids having a predicted molecular weight of 31kDa. Sequence analysis revealed that TgDPA had a deoxyribose phosphate aldolase (DeoC) domain with about 30% homology with its Escherichia coli counterpart. RT- and quantitative PCR analyses showed that the TgDPA gene was more expressed in bradyzoites and that its expression gradually increased during in vitro tachyzoite-to-bradyzoite stage conversion. A polyclonal antibody against recombinant TgDPA protein was raised in rabbits, and immunofluorescent analysis demonstrated that TgDPA was expressed in bradyzoites in vivo and in vitro. These findings indicate that the TgDPA gene is a new bradyzoite-specific marker and might play a role in bradyzoites.
Subject(s)
Aldehyde-Lyases/genetics , Gene Expression Regulation, Enzymologic , Toxoplasma/genetics , Aldehyde-Lyases/biosynthesis , Aldehyde-Lyases/chemistry , Amino Acid Sequence , Animals , Antibodies, Protozoan , Blotting, Western , Cloning, Molecular , Electrophoresis, Polyacrylamide Gel , Female , Fluorescent Antibody Technique, Indirect , Kinetics , Male , Mice , Mice, Inbred ICR , Microscopy, Confocal , Molecular Sequence Data , Polymerase Chain Reaction/methods , RNA, Messenger/analysis , Rabbits , Sequence Analysis , Toxoplasma/enzymology , Toxoplasma/immunologyABSTRACT
Toxoplasma gondii, unlike its mammalian host, utilizes a type II fatty acid biosynthesis pathway in which the steps of fatty acid biosynthesis are catalyzed by independent enzymes. Due to this difference, the enzymes of this pathway are good targets for the development of new therapeutic drugs directed against toxoplasmosis. In this report, we show by using reverse transcription-polymerase chain reaction analysis that beta-Hydroxyacyl-acyl carrier protein dehydratase (TgFABZ) is expressed both in tachyzoites and bradyzoites. Indirect immunofluorescence antibody test further shows the localization of TgFABZ protein in the apicoplast of both tachyzoites and bradyzoites. Enzyme dynamic analysis shows that the purified recombinant TgFABZ protein is soluble and active. The Km value of the enzyme for its substrate analog crotonoyl-CoA was estimated to be 82.57 +/- 10 microM.
Subject(s)
Hydro-Lyases/genetics , Hydro-Lyases/metabolism , Protozoan Proteins/genetics , Protozoan Proteins/metabolism , Toxoplasma/enzymology , Acyl Coenzyme A/metabolism , Animals , Antigens, Protozoan/analysis , Female , Fluorescent Antibody Technique, Indirect , Gene Expression Profiling , Hydro-Lyases/isolation & purification , Kinetics , Mice , Protozoan Proteins/isolation & purification , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Reverse Transcriptase Polymerase Chain Reaction/methods , Toxoplasma/chemistryABSTRACT
We developed a sandwich ELISA for the detection of circulating Toxoplasma gondii MIC10 antigens. In T. gondii culture supernatant, MIC10 was detected in a growth dependent manner. Mice were infected with a lethal dose of either a virulent RH strain, an avirulent Beverley strain or a sub-lethal dose of a PLK strain of T. gondii. MIC10 appeared 2 days after infection and increased gradually in the sera of RH-infected mice. A detectable but significantly lower amount of MIC10 was observed in the sera of mice infected intraperitoneally with Beverley tachyzoites. In contrast, the MIC10 antigen in mice sera following oral infection with Beverley cysts was below detectable levels during the course of the experiment. In sera of PLK-infected mice, MIC10 was predominantly observed between late acute and early chronic phase. Our data show that the kinetics of circulating MIC10 differs depending on the strain and route of infection.
Subject(s)
Antigens, Protozoan/blood , Toxoplasma/immunology , Toxoplasmosis, Animal/immunology , Animals , Antibodies, Protozoan/biosynthesis , Antibodies, Protozoan/immunology , Antigens, Protozoan/genetics , Blotting, Western , Electrophoresis, Polyacrylamide Gel , Enzyme-Linked Immunosorbent Assay/methods , Expressed Sequence Tags/chemistry , Female , Gene Expression Profiling , Kinetics , Male , Mice , Mice, Inbred BALB C , Protozoan Proteins/blood , Protozoan Proteins/genetics , Rabbits , Toxoplasma/genetics , Toxoplasma/pathogenicity , Toxoplasmosis, Animal/diagnosis , Toxoplasmosis, Animal/parasitology , VirulenceABSTRACT
A combination of antigenic regions of microneme proteins have been previously reported as being protective against chronic toxoplasmosis. In this work, we evaluated immune responses induced by immunizing BALB/c and C57BL/6 mice intradermally with plasmid DNA encoding the protein sequences of Toxoplasma gondii AMA1, MIC2, M2AP and BAG1. Mice immunized with the AMA1 gene developed high levels of serum IgG2a and c antibodies as well as cellular immune responses associated with IFN-gamma synthesis suggesting a modulated Th1 type of response. Immunization with the AMA1 gene resulted in a partial but significant protection against the acute phase of toxoplasmosis compared to MIC2, M2AP and BAG1 genes. Therefore, the AMA1 gene appears to generate a strong specific immune response and also provides effective protection against toxoplasmosis more than the MIC2, M2AP and BAG1 genes.
