ABSTRACT
High resolution two-dimensional separation of Toxoplasma gondii tachyzoite lysate revealed up to 224 distinct protein spots in Coomassie-stained gel. Computional matching of 14 digitized gels yielded a standard two-dimensional proteome map. The excretory T. gondii dense granule proteins GRA1-GRA8, S16/acid phosphatase, nucleoside triphosphate hydrolase, and H4 were identified by Western blotting of both total gel and isolated protein spots. In addition, two excretory antigens defined by parasite-specific monoclonal T cells, p36 and p40, were mapped by a novel T-cell blotting technique based on electroeluting single protein spots and testing the eluates for antigenic activity against the T-cell clones. In summary, these results represent a first step in Toxoplasma proteome analysis.
Subject(s)
Antigens, Protozoan/analysis , Proteome , Toxoplasma/chemistry , Animals , Antigens, Protozoan/immunology , Blotting, Western , CD4-Positive T-Lymphocytes/immunology , Electrophoresis, Gel, Two-Dimensional , Lymphocyte Activation , Peptide Mapping/methods , Protozoan Proteins/analysis , Toxoplasma/immunologyABSTRACT
Determinants of Toxoplasma gondii virulence are still unknown, although genetic markers associated with T. gondii pathogenicity or host susceptibility to infection have been identified. To define indicator proteins of mouse virulence, type I strain parasites were attenuated by continuous passage in fibroblast culture and compared with the parental strain passaged in mice. The loss of acute virulence, evident by a 1000-fold higher pathogen dose causing 100% lethality in mice correlated with a less efficient infection of inflammatory cells at the site of inoculation, while parasite proliferation and invasiveness in vitro proved unimpaired. Infection with the attenuated parasites elicited earlier local interleukin-12 and strong interferon-gamma responses in vivo, although the activity that triggers interleukin-12 secretion in macrophages is reduced in the attenuated compared to the virulent strain variant. The interleukin-12-inducing T. gondii stimulus was identified as a protein(s) present in tachyzoite excretory products. Comparative proteome analysis combined with immunodetection and quantitation of a variety of T. gondii antigens indicated that the steady-state levels of actin, catalase, microneme protein 5, as well as dense granule proteins 1, 2, 3, 4, 5, 7, 8 and nucleoside triphosphate hydrolase 1 are decreased in the attenuated phenotype, whereas the surface antigen 1 and rhoptry protein 1 are produced at a similar level by virulent and attenuated parasites. In conclusion, these findings reveal a correlation between the efficient establishment of T. gondii infection in vivo and parasite synthesis of actin, catalase and several excretory proteins, and thus postulate a role for these molecules in acute virulence.
Subject(s)
Interleukin-12/metabolism , Protozoan Proteins/metabolism , Toxoplasma/pathogenicity , Toxoplasmosis, Animal/parasitology , Actins/metabolism , Animals , Catalase/metabolism , Macrophages/immunology , Macrophages/metabolism , Mice , Mice, Inbred BALB C , Proteome , Protozoan Proteins/immunology , Toxoplasma/growth & development , VirulenceABSTRACT
Diagnosis of T. gondii invasions is based mainly on serological tests, because of their relative simplicity and availability. In the present work we examined efficiency of immunoblot and two inhouse ELISA assays in anti-Toxoplasma antibody detection, comparing our results with those obtained using commercial ELISA kit of Organon Teknika. We found that inhouse ELISAs and immunoblot can be helpful in diagnosing toxoplasmosis.
Subject(s)
Antibodies, Protozoan/blood , Antigens, Protozoan/analysis , Toxoplasma/immunology , Toxoplasmosis, Congenital/diagnosis , Toxoplasmosis, Congenital/parasitology , Adult , Animals , Biomarkers/blood , Enzyme-Linked Immunosorbent Assay/methods , Female , Humans , Immunoglobulin G/blood , Immunoglobulin M/blood , Infant , Infant, Newborn , Mice , Middle Aged , Pregnancy , Serologic Tests/methodsABSTRACT
The paper presents antigenic structure of T. gondii tachyzoites and bradyzoites in respect of potential use of some chosen antigens for diagnostic and immunoprophylactic purposes.
Subject(s)
Antigens, Protozoan/analysis , Protozoan Proteins/analysis , Toxoplasma/genetics , Toxoplasma/immunology , Animals , Antibodies, Protozoan/analysis , Antigens, Protozoan/classification , Cats , Fluorescent Antibody Technique, Indirect/methods , Host-Parasite Interactions/immunology , Humans , Life Cycle Stages/genetics , Oocysts/immunology , Protozoan Proteins/classification , Protozoan Vaccines/therapeutic use , Toxoplasma/parasitology , Toxoplasmosis/immunology , Toxoplasmosis/parasitology , Toxoplasmosis, Animal/immunology , Toxoplasmosis, Animal/parasitologyABSTRACT
The paper presents chosen experimental strategies for identifying of Toxoplasma gondii antigens recognized by specific T lymphocytes.
Subject(s)
Antigens, Protozoan/isolation & purification , Immunoglobulin G/analysis , Immunoglobulin M/analysis , T-Lymphocytes/immunology , Toxoplasma/immunology , Animals , Antibodies/immunology , Antibody Formation/immunology , Antigens, Protozoan/immunology , Electrophoresis, Polyacrylamide Gel/methods , Epitopes/biosynthesis , Epitopes/immunology , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , Toxoplasma/parasitology , Toxoplasmosis/immunology , Toxoplasmosis/parasitologyABSTRACT
The paper presents antigenic structure of T. gondii tachyzoites and bradyzoites in respect of potential use of some chosen antigens for diagnostic and immunoprophylactic purposes.
