title: Identification and characterization of an immunogenic antigen, enolase 2, among excretory/secretory antigens (ESA) of Toxoplasma gondii.
abstract: An immunogenic protein, enolase 2, was identified among the secreted excretory/secretory antigens (ESAs) from Toxoplasma gondii strain RH using immunoproteomics based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Enolase 2 was cloned, sequenced, and heterologously expressed. BLAST analysis revealed 75-96% similarity with enolases from other parasites. Immunoblotting demonstrated good immunoreactivity of recombinant T. gondii enolase (Tg-enolase 2) to T. gondii-infected animal serum. Purified Tg-enolase 2 was found to catalyze dehydration of 2-phospho-d-glycerate to phosphoenolpyruvate. In vitro studies revealed maximal activity at pH 7.5 and 37 °C, and activity was inhibited by K(+), Ni(2+), Al(3+), Na(+), Cu(2+) and Cr(3+). A monoclonal antibody against Tg-enolase 2 was prepared, 1D6, with the isotype IgG2a/κ. Western blotting revealed that 1D6 reacts with Tg-enolase 2 and native enolase 2, present among T. gondii ESAs. The indirect immunofluorescence assays showed that enolase 2 could be specifically detected on the growing T. gondii tachyzoites. Immunoelectron microscopy revealed the surface and intracellular locations of enolase 2 on T. gondii cells. In conclusion, our results clearly show that the enzymatic activity of T. gondii enolase 2 is ion dependent and that it could be influenced by environmental factors. We also provide evidence that enolase 2 is an important immunogenic protein of ESAs from T. gondii and that it is a surface-exposed protein with strong antigenicity and immunogenicity. Our findings indicate that enolase 2 could play important roles in metabolism, immunogenicity and pathogenicity and that it may serve as a novel drug target and candidate vaccine against T. gondii infection.
authors.affiliation: Chinese Academy of Agricultural Sciences, Shanghai Veterinary Research Institute, CAAS, 518 Ziyue Road, Minhang, Shanghai 200241, PR China.
authors.forename: Wei
authors.initials: W
authors.lastname: Jiang
authors.affiliation: Chinese Academy of Agricultural Sciences, Shanghai Veterinary Research Institute, CAAS, 518 Ziyue Road, Minhang, Shanghai 200241, PR China; College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, PR China.
authors.forename: Jun-Xin
authors.initials: JX
authors.lastname: Xue
authors.affiliation: Chinese Academy of Agricultural Sciences, Shanghai Veterinary Research Institute, CAAS, 518 Ziyue Road, Minhang, Shanghai 200241, PR China.
authors.forename: Ying-Chun
authors.initials: YC
authors.lastname: Liu
authors.affiliation: Chinese Academy of Agricultural Sciences, Shanghai Veterinary Research Institute, CAAS, 518 Ziyue Road, Minhang, Shanghai 200241, PR China.
authors.forename: Tao
authors.initials: T
authors.lastname: Li
authors.affiliation: Chinese Academy of Agricultural Sciences, Shanghai Veterinary Research Institute, CAAS, 518 Ziyue Road, Minhang, Shanghai 200241, PR China.
authors.forename: Xian-Gan
authors.initials: XG
authors.lastname: Han
authors.affiliation: Chinese Academy of Agricultural Sciences, Shanghai Veterinary Research Institute, CAAS, 518 Ziyue Road, Minhang, Shanghai 200241, PR China.
authors.forename: Shao-Hui
authors.initials: SH
authors.lastname: Wang
authors.affiliation: Chinese Academy of Agricultural Sciences, Shanghai Veterinary Research Institute, CAAS, 518 Ziyue Road, Minhang, Shanghai 200241, PR China.
authors.forename: Yong-Jun
authors.initials: YJ
authors.lastname: Chen
authors.affiliation: Chinese Academy of Agricultural Sciences, Shanghai Veterinary Research Institute, CAAS, 518 Ziyue Road, Minhang, Shanghai 200241, PR China.
authors.forename: Jingjing
authors.initials: J
authors.lastname: Qi
authors.affiliation: Chinese Academy of Agricultural Sciences, Shanghai Veterinary Research Institute, CAAS, 518 Ziyue Road, Minhang, Shanghai 200241, PR China. Electronic address: yus@shvri.ac.cn.
authors.forename: Sheng-Qing
authors.initials: SQ
authors.lastname: Yu
authors.affiliation: Chinese Academy of Agricultural Sciences, Shanghai Veterinary Research Institute, CAAS, 518 Ziyue Road, Minhang, Shanghai 200241, PR China. Electronic address: wangquan@shvri.ac.cn.
authors.forename: Quan
authors.initials: Q
authors.lastname: Wang
mesh_terms: D000818:Animals; D058846:Antibodies, Monoclonal, Murine-Derived / Q000276:immunology*; D000913:Antibodies, Protozoan / Q000276:immunology*; D000953:Antigens, Protozoan / Q000096:biosynthesis / Q000276:immunology / Q000302:isolation & purification / Q000494:pharmacology; D004926:Escherichia coli / Q000378:metabolism; D007114:Immunization; D051379:Mice; D008807:Mice, Inbred BALB C; D010751:Phosphopyruvate Hydratase / Q000096:biosynthesis / Q000276:immunology / Q000302:isolation & purification / Q000494:pharmacology; D015800:Protozoan Proteins / Q000096:biosynthesis / Q000276:immunology / Q000302:isolation & purification / Q000494:pharmacology; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000276:immunology / Q000302:isolation & purification / Q000494:pharmacology; D014122:Toxoplasma / Q000201:enzymology / Q000276:immunology
chemical_list: D058846:Antibodies, Monoclonal, Murine-Derived; D000913:Antibodies, Protozoan; D000953:Antigens, Protozoan; D015800:Protozoan Proteins; D011994:Recombinant Proteins; D010751:Phosphopyruvate Hydratase
keywords: Enolase 2; Enzyme activity; Excretory/secretory antigens; Immunoproteomic; Toxoplasma gondii