Characterization of an excreted/secreted antigen form of 14-3-3 protein in Toxoplasma gondii tachyzoites (original) (raw)

Ultrastructural and biochemical studies on the immunohistochemistry of Toxoplasma gondii antigens using monoclonal antibodies

Histochemistry, 1983

To determine the cellular distribution of Toxoplasma antigens, RH strain tachyzoites were incubated with either one of three monoclonal antibodies (FMC 19, FMC 20, FMC 22) to T. gondii, or one of two controls (the murine myeloma protein MOPC 21, or phosphate buffered saline), and then incubated with peroxidase-labelled goat-antimouse IgG. Diaminobenzidine was added as substrate and electron microscopy was used to localize the reaction. All three antibodies bound to the entire periphery of the tachyzoite surface membrane. To ascertain the chemical composition of the antigens against which seven monoclonal antibodies (FMC 18, FMC 19, FMC 20, FMC 22, FMC 23, 2Gli, 3E6) to T. gondii reacted, untreated, pronase-treated, or periodate-treated tachyzoites were incubated with the antibodies or MOPC 21, and then with 112511-Protein A. The pronase-treated tachyzoites showed reduced binding for six of the antibodies, compared with the reduction in binding of MOPC 21 with the pronase-treated parasites. The periodate-treated tachyzoites had reduced binding for FMC 18 only. The results of these experiments confirm that most Toxoplasma surface antigens are protein in nature, and are consistent with the hypothesis that at least one cytoplasmic antigen is secreted onto the parasite cell surface.

Electrophoretic Patterns of Toxoplasma gondii Excreted/Secreted Antigens and Their Role in Induction of the Humoral Immune Response

Jundishapur Journal of Microbiology, 2014

Toxoplasma gondii is an obligatory intracellular protozoan parasite on which studies are pending regarding production of vaccine. To date, the production of human vaccine has not been successful where approximately one third of the world's population is thought to be infected with T. gondii. The present study was designed to compare the electrophoretic patterns of T. gondii excreted/secreted antigens (ESAs) and determine their role in the stimulation of the humoral immune response. T. gondii ESAs were prepared from cell cultures (albino rat fibroblast) and cell-free mediums (RPMI-1640). Next, the SDS-PAGE technique was used for comparing molecular weights of the antigens. Forty C57BL/6 mice were divided randomly into four groups (n = 10). Immunization was performed subcutaneously at an interval of 2 weeks in two groups by injecting 100 µg of each of the above-mentioned antigens. Two groups, as negative control, also received fibroblast lysate proteins or adjuvant separately. All of the groups were then challenged with the T. gondii RH strain. Serum samples were collected from all mice and measured by immunoblotting technique for detection of immunogenic antigens. The electrophoretic mobility of the prepared antigens/proteins from cell culture, cell-free media, Fibroblast Lysate Proteins and Toxoplasma Lysate Antigens (TLA) showed 13, 12, 8 and 8 bands, respectively. The case groups, in challenge with T. gondii (RH strain), showed more survival prolongation than the control groups. Furthermore, the survival period was identical for both case groups with a tendency for slightly higher survival of mice receiving ESA from cell-free medium. Analysis of sera by immunoblotting also revealed one band of 65 KDa in sera from both case groups. We suggest that this band be extracted and its amino acids sequence determined to produce Synthetic Polypeptide for immunization studies.

Detection and characterization of membrane antigens of Toxoplasma gondii

Journal of Immunology, 1980

Toxoplasma gondii tachyzoites were surface radioiodinated by the lactoperoxidase technique, and the solubilized membrane proteins were analyzed by two-dimensional polyacrylamide gel electrophoresis. Four major labeled proteins with apparent m.w. of 43,000, 36,000, 27,000, and 14,000 were detected. None of the radioiodinated proteins bound to concanavalin A-Sepharose. When a panel of eight different fluorescein-conjugated lectins was used in an attempt to characterize further the nature of the cell membrane, none of the lectins bound to intact tachyzoites. Two-dimensional polyacrylamide gel electrophoresis did not reveal any significant differences among three different strains of Toxoplasma. Each of the radioiodinated surface proteins was precipitable by sera from mice chronically infected with the same strain as well as by a series of sera from mice infected with other strains. Sera from humans with acute Toxoplasma infection showed more variability in that some precipitated all labeled proteins whereas others precipitated only two or three of them. Monoclonal antibodies (2611 and 3E6) prepared by hybridization of spleen cells from Toxoplasma-immune mice with myeloma cells consistently precipitated both the solubilized 35,000 and 14,000 dalton proteins, whereas 1E3 precipitated the 43,000-dalton protein and l E l l the 27,000-dalton protein. Toxoplasma gondii is a ubiquitous intracellular protozoan parasite that is capable of infecting all species of mammals (1). Serologic studies in humans indicate that from 20 to 90% of adult populations have been infected with Toxoplasma (2-4), and serious disease due to this parasite occurs in congenitally infected children and in immunosuppressed patients. In veter

Molecular characterization of a 65-kilodalton Toxoplasma gondii antigen expressed abundantly in the matrix of tissue cysts

Molecular and Biochemical Parasitology, 1994

We describe the cloning and characterization of a novel antigen expressed in the bradyzoite stage of Toxoplasma gondii. A cDNA library was constructed in bacteriophage Agtll Sfi-Not using messenger RNA molecules isolated from cysts of the ME49 strain of T. gondii. The recombinant phage library was subjected to screening with polyclonal antibodies against bradyzoite antigens. This screening identified a recombinant antigen that was recognized strongly by polyclonal antibodies against bradyzoite antigens as well as by sera from mice chronically infected with T. gondii. The native antigen is a protein of 65 kDa that localized to the matrix of the cyst and the cyst wall surrounding the bradyzoites. The antigen was found to be expressed abundantly in cysts but could not be detected in tachyzoites or within the parasitophorous vacuole of tachyzoite infected host cells. Genomic and cDNA sequence of the gene revealed an open reading frame encoding 452 amino acids interrupted by 2 introns: a 503-bp intron located in the 5' untranslated region preceding the protein coding sequence and a ll0-bp intron located 95 bp downstream of the first ATG.

Toxoplasma gondii excretory secretory antigenic proteins of diagnostic potential

APMIS, 2012

Infection with Toxoplasma gondii is widespread and important in humans, especially pregnant women and immunosuppressed patients. A panel of tests is usually required for diagnosis toxoplasmosis. Excretory secretory antigen (ESA) is highly immunogenic, and thus it is a good candidate for investigation into new infection markers. ESA was prepared from tachyzoites of RH strain of T. gondii by mice intraperitoneal infection. Sera were obtained from several categories of individuals who differed in their status of anti-Toxoplasma IgM, IgG and IgG avidity antibodies. The ESA was subjected to SDS-PAGE, two-dimensional gel electrophoresis and Western blot analysis. Antigenic bands of approximate molecular weights of 12, 20 and 30 kDa, when probed with anti-human IgM-HRP and IgA-HRP, showed good potential as infection markers. The highest sensitivity of the bands was 98.7% with combination of IgM and IgA blots with sera of patients with anti-Toxoplasma IgM+ IgG+. The specificities were 84% and 70% with sera from other infections and healthy controls in IgM blots and IgA blots respectively. By mass spectrometry, the 12 kDa protein was identified as thioredoxin. The two top proteins identified for 20 kDa molecule were microneme protein 10 and dense granule protein 7; whereas that for 30 kDa were phosphoglycerate mutase 1 and phosphoglycerate mutase.

Isolation and characterization of toxoplasma exo-antigens from In vitro culture in MRC5 and vero cells

International Journal for Parasitology, 1987

Isolation and characterization of toxoplasma exo-antigens from in vitro culture in MRCS and VERO cells. International Journalfor Parasitology 17: 829-834. Exo-antigens from the supernatant medium of in vitro cultures of Toxoplasmsa gondii on Vero and MRC5 cells have been characterized by different immunological techniques: enzyme-linked immunosorbent assay (ELISA), counter immunoelectrophoresis (CE), twodimensional immunoelectrophoresis (2-DIEP) and gel electrophoresis of derivated ELISA (GEDELISA). These exo-antigens are similar in molecular sizes, thermostability and of proteic nature with glucidic components. Their enzymatic properties and precipitation arcs differ. The exo-antigens, produced on Vero cells (monkey kidney cells), seem to be more antigenic than those produced on MRCS cells (human foetal lung cells). The formaldehyde treatment stabilizes the exo-antigens, in spite of eliminating certain epitopes, and increases their migration rate; it has practically no effect on their enzymatic or antigenic activities. The pl of the formolated exo-antigens produced on MRCS cells are mainly under 5.06.

Generation of antibodies againstToxoplasma gondii antigen associated with dense granules and the parasitophorous vacuole of the host cell

Parasitology Research, 1992

Several reports indicate that Toxoplasma antigens may occur in the blood during acute T. gondii infection (for references see Asai et al. 1987; Hassl et al. 1988; Huskinson et al. 1989), but the characteristics of circulating antigens have not yet been defined in detail and reagents have not yet been developed for diagnostic assays. As the first step in an attempt to demonstrate circulating T. gondii antigens, we investigated the generation of monoclonal anti-T, gondii antibodies by immunizing mice with plasma from T. gondii-infected mice. Blood from BALB/c mice that had been killed at 4 days after intraperitoneal infection with 2 x 104 T. gondii RH-strain parasites was collected into heparinized tubes. After the tubes had been centrifuged at 1,000 g for 10 min, the plasma was filtered through a 0.45-~tm membrane (Millipore Corporation, USA), and 0.15 ml filtered plasma supplemented with Freund's incomplete adjuvant was injected intraperitoneally into BALB/c mice. Two 0.1-ml booster injections were given intravascularly on days 9 and 40. The last injection was given 4 days before the immunized animals were killed. Hybridomas were obtained by the fusion of spleen cells from immunized animals with SP2 lymphoma cells (Shulman et al. 1978). Antibody-secreting hybridome cell clones were identified by subjecting supernatant fluids to the indirect fluorescent antibody test (IFAT; Linder et al. 1987). The antigen used in the screening assay was derived from T. gondii parasites that had been fixed on microscope slides in acetone at-20~ for 10 min, Using different fixation protocols, we could differentiate between antibodies directed against exposed parasite antigens and those directed against hidden antigens. To identify antibody reactivity with surface antigens of intact parasites, we carried out the IFAT using live Toxoplasma or parasites fixed in 3.5% paraformaldehyde as antigen sources. To make intracellular antigens accessible, fixed parasites were made permeable by incubation in detergent solution consisting of 0.1% Nonidet-Offprint requests to: E. Linder

Toxoplasma gondii: Effect of infection on expression of 14-3-3 proteins in human epithelial cells

Experimental Parasitology, 2008

14-3-3 Proteins are expressed in most eukaryotes organisms and play varied and crucial roles in a wide range of regulatory processes. In mammalian cells, seven 14-3-3 isoforms have been identified. However, it is not known what effect infection has on 14-3-3 isoform expression. In this study human colonic carcinoma cell lines were infected with Toxoplasma gondii for 24 h and expression of 14-3-3 proteins was determined by RT-PCR. HT-29 cells only expressed 3 out of the 7 isoforms while 5 and all 7 isoforms were found in HCT-116 and Caco-2 cells, respectively. Infection had little or no effect in the expression of 14-3-3c, e, r, and n; but in HCT-116 cells induced expression of 14-3-3g and r, while 14-3-3b, g, and n were induced in HT-29 cells. If 14-3-3 proteins are involved in cell survival and/or prevention of parasite replication, longer incubation times may be required as no differences in percentage of infection were found among the cell lines at 24 h post-infection.

Analysis of Toxoplasma gondii antigens with sera from toxoplasmosis patients

Revista da Sociedade Brasileira de Medicina Tropical, 1998

Some proteins of the Toxoplasma gondii are recognized by IgG, IgM and IgA antibodies in patients with acute and chronic toxoplasmosis, depending on the strain and stage of the Toxoplasma. Sixty-nine sera from immunocompetent individuals were studied through the Western-Blot Test: 20 has an acute infection, 29 has a chronic toxoplasmosis infection and 20 were healthy (seronegatives). The protein analysis revealed by IgG and IgM antibodies were performed through the Immunoplot method in order to know their recognition frequency (f) and be valued as infection markers. In the acute phase, the IgM antibodies showed a recognition frequency (f = 0.60) for the 60kDa protein, and in the chronic phase the IgG antibodies showed a recognition frequency (f = 0.68) for the 12kDa protein. Seronegatives revealed no type of band. The protein of 12kDa can be a diagnostic marker of the chronic phase while protein 60kDa of the acute phase of toxoplasmosis.

Comparison of three forms of antigens in the demonstration of cell-mediated immune response in murine toxoplasmosis

Biochemical and Biophysical Research Communications, 1992

Mice were chronically infected with cysts of ME 49 strain of Toxoplasmagondii. At different periods post-infection, their spleens were removed and single cell suspensions were made. Lymphocyte transformation experiments were performed on the lymphocyte suspensions using threedifferent kinds of antigensof ME49strain of T. gondii, namely soluble, excretory/secretory and cystic forms. The results showed that the pattern of lymphocyte responsiveness wasdependenton thekindofantigenemployedforinductionofthe blastogenesis. Using soluble and cystic forms of the antigen, different periods of lymphocyte suppression and lymphocyte proliferation were demonstrated. However, with the use of excretory/secretory antigen, no significant suppression of lymphocyte stimulation was noted throughout the course of infection. Thus excretory/secretory antigen may be the best form of antigen for stimulation of the cell-mediated immune response and hence it appears to be a good candidate for vaccine in toxoplasmosis.