Identification of Toxoplasma gondii SUB1 Antigen as a Marker for Acute Infection by Use of an Innovative Evaluation Method

Abstract

By the separation of Toxoplasma lysate using two-dimensional gel electrophoresis and its analysis with human serum samples and mass spectrometry, the subtilisin-like protein (SUB1) was identified to be a potential marker for acute toxoplasmosis. Following expression of the C-terminal domain of SUB1 in Escherichia coli, it was tested in a line blot assay using a total of 80 human serum samples. Two computer programs based on different evaluation strategies were used for judgment of the line blot results: (i) a time-dependent method with a predefined cutoff value and (ii) a fixed-time-point method with a calculated cutoff. Thereby, SUB1 was proven to be rather reactive with specific immunoglobulin A (IgA), IgM, and IgG of patients with an acute infection. This finding makes this antigen an attractive candidate for improving diagnosis of toxoplasmosis and demonstrates that not only the selection of respective antigens but also the evaluation method chosen are important for the evaluation of new diagnostic markers.One of the most successful protozoan parasites is Toxoplasma gondii. In humans and other intermediate hosts, the tachyzoite stage of the parasite represents the active phase of infection, whereas the bradyzoite stage represents the chronic or latent phase of infection. In immunocompetent human hosts, the infection is asymptomatic in most cases. A special threat exists when primary infection takes place in the developing fetus or in immunocompromised hosts, particularly patients suffering from AIDS (21, 27) and transplant recipients (6, 27).Many commercial tests are available for the serological diagnosis of T. gondii infection. Here, the enzyme-linked immunosorbent assay (ELISA) and immunoblotting are widespread techniques that are performed automatically or manually for the detection of T. gondii-specific immunoglobulin G (IgG), IgM, and IgA.We tried to identify Toxoplasma antigens that might be suitable for the serological identification of early infection by twodimensional (2D) gel electrophoresis and mass spectrometry (MS) analysis. After recombinant production of the IgA-reactive T. gondii protein subtilisin-like protein 1 (SUB1), we evaluated it on human serum samples which were previously tested with commercial tests and classified as having either no T. gondii infection, acute infection (high IgM or IgA titer), or chronic infection (no IgM or IgA, high IgG titer). MATERIALS AND METHODSParasites and host cells. Tachyzoites of the type II strain NTE were isolated from an L929 fibroblast-T. gondii coculture. These parasites were used to infect human foreskin fibroblasts (HFFs) and cultured in Dulbecco modified Eagle medium containing 10% fetal calf serum, 1% penicillin-streptomycin, 1% sodium pyruvate, and 1% nonessential amino acids (NEAA) for 3 to 4 days. Infected cells were cultured at 37°C in 5% CO 2 atmosphere.T. gondii antigen preparation. Extracellular parasites were isolated from the supernatant of infected HFFs by filtration (pore size, 0.02 m; Millipore, Billerica, MA). The cell pellet of 4 ...

Construction and Sequencing of Dense Granular14 (GRA14) Gene of Toxoplasma gondii (RH) in Expression Prokaryotic Plasmid PET32a: A Preliminary Study in Vaccine Production

“…The C-terminal fragment of the SUB1 gene (nucleotide positions 1657 to 2316) on the basis of the antigenic index containing amino acid residues 552 to 777 was prepared by PCR amplification and then 660 bp PCR product was cloned into pQE-30 expression vector. 47 In the performed study, the 1227 bp fragment of T. gondii GRA14 gene was cloned into pET32a expression plasmid and ready to produce protein and using of that in diagnostic studies, recombinant vaccine production and immunologic surveys in animal models.…”

Cloning and Sequencing of Truncated Toxoplasma gondii Subtilisin-Like 1 Antigen

“…Hruzik et al introduced Cterminal domain of TgSUB1 as a potential marker for serodiagnosis of acute toxoplasmosis. They suggested this recombinant protein can be used with other recombinant antigens such as SAG1 for diagnosis of infection [34].…”

“…TgSUB1 is a GPI-anchored micronemal protease that is first released onto the surface of parasites during invasion in a calcium-dependent manner and then shed into the media with other micronemal proteins. [34]. SUB1 is also studied in other genus of parasites, like Neospora caninum, It may be assumed that cross-reactions may occur with their closely related parasite T. gondii if such antigens applied in serological diagnosis.…”

Identification and characterization of peptidases secreted by quahog parasite unknown (QPX), the protistan parasite of hard clams

“…Secreted C1 peptidases also determine pathogenesis of Enteamoeba histolytica, which uses these enzymes to digest the extracellular matrix of human tissue in cluding collagen, elastin, fibrinogen, and laminin as well as immu noglobulin A (Que & Reed 2000). The S8 family, or subtilisins, are universal virulence factors of many pathogenic bacteria (Brown et al 2000, Kennan et al 2010, Bonifait et al 2011, protozoa (Miller et al 2001, Jean et al 2003, Wanyiri et al 2009, Swenerton et al 2010, Hruzik et al 2011, and fungi (Moser et al 1994, Huang et al 2004, Withers-Martinez et al 2004. Subtilisins are also secreted by Perkinsus marinus, a protozoan pathogen of oysters, and are considered as important components of its virulence capabilities (Earnhart et al 2004).…”