Toxoplasma gondii excretory secretory antigenic proteins of diagnostic potential (original) (raw)
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Recombinant Dense Granular Protein (GRA5) for Detection of Human Toxoplasmosis by Western Blot
BioMed Research International, 2014
Toxoplasma gondiiinfects all warm-blooded animals, including humans, causing serious public health problems and great economic loss for the food industry. Commonly used serological tests require costly and hazardous preparation of wholeToxoplasmalysate antigens from tachyzoites. Here, we have evaluated an alternative method for antigen production, which involved a prokaryotic expression system. Specifically, we expressedT. gondiidense granular protein-5 (GRA5) inEscherichia coliand isolated it by affinity purification. The serodiagnostic potential of the purified recombinant GRA5 (rGRA5) was tested through Western blot analysis against 212 human patient serum samples. We found that rGRA5 protein was 100% specific for analysis of toxoplasmosis-negative human sera. Also, rGRA5 was able to detect acute and chronicT. gondiiinfections (sensitivities of 46.8% and 61.2%, resp.).
In the present study, we applied the combination of one-dimensional gel electrophoresis, immunoblot and nano-liquid chromatography-tandem mass spectrometry (nanoLC-MS/MS) to identify potential immunogenic proteins of Toxoplasma gondii tachyzoites that can be used for the development of reliable assays in the serodiagnosis of acquired toxoplasmosis in immunocompetent subjects. For this purpose, we developed an immunoblot using soluble and membrane extracts of GT1 Toxoplasma gondii tachyzoites and tested 194 positive and 100 negative sera obtained from pregnant women. Five bands of soluble antigens (98 kDa, 36 kDa, 33 kDa, 32 kDa and 21 kDa) and 4 bands of membrane antigens (41 kDa, 35 kDa, 32 kDa and 30 kDa) were selected as the most valuable in terms of sensitivity and specificity. Among these bands, only 2 bands of soluble antigen (33 kDa and 32 kDa) and 2 bands of membrane antigen (32 kDa and 30 kDa) showed a specificity ≥ 90%. After mass spectrometry and bioinformatics analysis, 7 proteins were identified as potential markers for serodiagnosis of toxoplasmosis. These proteins are: SRS34A, GRA7, GRA1, DG32, MIC5, ROP5 and Toxofilin. These proteins showed a 86% to 100% homology with proteins of both VEG and ME49 strains of T. gondii and a 58% to 87% homology with Hammondia hammondi; and can be considered as attractive candidates for the development of an immunochromatography test that can be used for the rapid diagnosis of toxoplasmosis and as a confirmatory test when routine techniques give equivocal results.
Diagnostic Microbiology and Infectious Disease, 2007
GRA2 is a highly immunogenic protein secreted from the dense granules of Toxoplasma gondii. Recent success in purifying full-length, soluble GRA2 from bacteria as a thioredoxin (TRX)-(Hisx6) fusion protein led to investigate the antigenicity of the recombinant protein against human sera. On immunoblots, TRX-(Hisx6)-GRA2 was recognized by sera collected in Iran from T. gondii-infected pregnant women. An IgG enzyme-linked immunosorbent assay was developed to evaluate the reactivity of sera, collected from pregnant women both in France and Iran, to the TRX-(Hisx6)-GRA2 fusion protein. Specificity of the test was 96.4%. Sensitivity of the GRA2 enzyme-linked immunosorbent assay ranged from 95.8% (sera collected in France) to 100% (sera collected in Iran) for sera of acute infection and from 65.7% (sera collected in France) to 71.4% (sera collected in Iran) for sera of chronic infection. The recombinant GRA2 could thus advantageously complement previously described T. gondii antigens for the serodiagnosis of acute Toxoplasma infection. D
Parasitology Research, 2012
Toxoplasma gondii infects all warm-blooded animals including humans, causing serious public health problems and great economic loss in the food industry. Commonly used serological tests involve preparation of whole Toxoplasma lysate antigens from tachyzoites which are costly and hazardous. An alternative method for better antigen production involving the prokaryotic expression system was therefore used in this study. Recombinant dense granular protein, GRA2, was successfully cloned, expressed, and purified in Escherichia coli, BL21 (DE3) pLysS. The potential of this purified antigen for diagnosis of human infections was evaluated through western blot analysis against 100 human serum samples. Results showed that the rGRA2 protein has 100 and 61.5 % sensitivity towards acute and chronic infection, respectively, in T. gondii-infected humans, indicating that this protein is useful in differentiating present and past infections. Therefore, it is suitable to be used as a sensitive and specific molecular marker for the serodiagnosis of Toxoplasma infection in both humans and animals.
BMC Structural Biology, 2012
Background: Toxoplasma gondii is an intracellular coccidian parasite that causes toxoplasmosis. It was estimated that more than one third of the world population is infected by T. gondii, and the disease is critical in fetuses and immunosuppressed patients. Thus, early detection is crucial for disease diagnosis and therapy. However, the current available toxoplasmosis diagnostic tests vary in their accuracy and the better ones are costly. Results: An earlier published work discovered a highly antigenic 12 kDa excretory-secretory (ES) protein of T. gondii which may potentially be used for the development of an antigen detection test for toxoplasmosis. However, the three-dimensional structure of the protein is unknown. Since epitope identification is important prior to designing of a specific antibody for an antigen-detection based diagnostic test, the structural elucidation of this protein is essential. In this study, we constructed a three dimensional model of the 12 kDa ES protein. The built structure possesses a thioredoxin backbone which consists of four α-helices flanking five β-strands at the center. Three potential epitopes (6-8 residues) which can be combined into one "single" epitope have been identified from the built structure as the most potential antibody binding site. Conclusion: Together with specific antibody design, this work could contribute towards future development of an antigen detection test for toxoplasmosis.
Zentralblatt für Bakteriologie, Mikrobiologie und Hygiene. Series A: Medical Microbiology, Infectious Diseases, Virology, Parasitology, 1987
The present study was performed to demonstrate circulating antigen (cag) of Toxo plasma gondii in the sera of orall y and intraperitone ally infected rabbit s and swine, to determ ine the time of their app earance after infection, and to characterize the antigenic compo nents of the cag by means of affinity chromatography, SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and electrophoretic blot onto nitrocellul ose sheets. Cag, as detected by an ind irect ELISA, was found in the sera of both rabb its and swine from week 5 to week 8 after infection. Electrophoretic separation of cag extracted from swine and human sera showed 5 and 8 distinct protein bands, respectively, the molecular weight of which ranged between 25 and 100 kD. After Western blot, 2 of the 5 protein bands of swine-cag (27 and 57 kD) and 3 of the 8 prote in band s of human cag (27, 32, and 57 kD) reacted with the ant i-Toxoplasma antibody used in the ELISA. On the basis of the data presented , the influence of the dose and mode of infection as well as that of the preparation method of antisera on cag detect ion is discussed. Zusammenfassung Zirkulier endes Antigen von Toxo plasma gondii (zAg) wurde in den Seren experimentell oral und intraperitoneal infizierter Kaninchen und Schweine mittels eines indirekten ELISA nac hgewiesen und der Zeitraum des Auftretens nach der Infektion festgestellt. Dariiber hina us wurde zAg aus den Schweineseren und einem Humanserum mittels Affinitatschromarographie isoliert und das Molekulargewicht der Proteine durch SDS-Polyacrylamid-Gel-Elektrophorese (SDS-PAGE) und Western Blot ermittelt. ZAg konnte in Schweine-und Kaninchen seren von der 5. bis zur 8. Woche nach der Infektion nachgewiesen werden. Durch elektrophoretische Auftrennung des zAg au s Schweine-und Humanseren in der SDS-PAGE konnten 5 bzw. 8 deutliche Proteinband en mit Molekulargewichten zwischen 25 und 100 kD identifiziert werden. Na ch der Transferierung auf Nitrozellulose reagierten 2 der Banden (MG 27 und 57 kD) des zAg aus Schweineseren und 3 Band en (MG 27, 32 und 57 kD) des zAg aus Humanserum mit dem im
African Journal of Microbiology Research, 2013
Toxoplasma gondii is the causative protozoan of toxoplasmosis which has a worldwide distribution among humans and warm-blooded animals. The diagnosis generally depends on serologic tests but the persistently high Immunoglobulin M (IgM) or low IgG avidity complicate diagnosis. It is essential to identify acute-stage-specific antigen to use in a single test for the definitive diagnosis of the acute disease, especially in pregnant women and immunocompromised individuals. In this study, we investigated the recognition of proteins of lysate antigen (LA) and excretory-secretory antigen (ESA) fractions of the virulent RH strain of T. gondii by the sera of patients who had recent or past infections and the sera of experimentally infected mice. As a result, no specific bands for discrimination of recent infection from the past one could be detected in our study but the results confirmed that proteins of approximately 20, 30 and 40 kDa were among the major targets for IgG responses during acute and chronic infection with T. gondii in humans.
Comprehensive Proteome Analysis of the Excretory/Secretory Proteins of Toxoplasma gondii
Bulletin of the Korean Chemical Society
Proteomic analyses of the excretory/secretory proteins from the RH strain of Toxoplasma gondii have been performed to understand their functions in the host-parasite interaction. A total of 34 proteins were identified from LC/MS/MS analysis and their abundance was estimated by spectral counting methods. Among them, 8 species of micronemal proteins (MICs), 2 species of rhoptry proteins (ROPs), and 6 species of dense granular proteins (GRAs) were confirmed. Besides these, 18 species of protein were newly identified, and their cellular functions were estimated from sequence analysis. The three most abundant of the 34 identified extractor/ secretory proteins−GRA1, GRA7 and GRA2−were confirmed to be highly expressed in T. gondii using the spectral count method. This phenomenon is another demonstration of the importance of GRA proteins for the penetration and survival of T. gondii.
Braun-2013-A Toxoplasma dense granule protein
Abbreviations used: BMDM, BMderived macrophage; DG, dense granule; HFF, human foreskin fibroblast; KIM, ki nase interacting motif; MOI, multiplicity of infection; PV, parasitophorous vacuole; PVM, PV membrane. A. Bougdour and M.A. Hakimi contributed equally to this paper.
2010
Toxoplasma gondii is an intracellular parasite of world-wide distribution. Infection in pregnant women may result in abortion and foetal abnormalities, and may be life-threatening in immunocompromised hosts. Many studies have been performed on developing an antigen detection assay for T. gondii. However, such assay is unavailable commercially, one reason may be due to lack of good data regarding Toxoplasma circulating antigens in the blood of actively infected individuals. Proteomics has greatly aided in discovery of disease infection markers for diagnostic purposes, thus this approach was used in this study to identify potential serum infection markers in toxoplasmosis. Initially, an ELISA employing monoclonal anti-Toxoplasma SAG1 (p30) as the capture antibody to detect T. gondii circulating antigens in serum samples was developed. Subsequently a study on protein profiles of serum from Toxoplasma gondii infection using two dimensional electrophoresis (2-DE) was performed, followed ...