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Characterization at the Molecular Level using Robust Biochemical Approaches of a New Kinase Protein
Published on: June 30, 2019
Expression and immunological characteristics of the surface-localized pyruvate kinase in Mycoplasma gallisepticum
Suibin He1, Jingjing Qi2, Shengqing Yu2
1Shanghai Veterinary Research Institute, the Chinese Academy of Agricultural Sciences (CAAS), 518 Ziyue Road, Shanghai 200241, PR China; Key Laboratory of Animal Disease Diagnosis & Immunology, College of Veterinary Medicine, Nanjing Agricultural University, No. 1 Weigang, Nanjing 210095, PR China.
Abstract:
The widespread avian pathogen Mycoplasma gallisepticum is a causative agent of respiratory disease. The wall-less prokaryotes lack some tricarboxylic acid cycle enzymes, therefore, the glycolysis metabolic pathway is of great importance to these organisms. Pyruvate kinase (PK) is one of the key enzymes of the glycolytic pathway, and its immunological characteristics in Mycoplasma are not well known. In this study, the M. gallisepticum pyruvate kinase fusion protein (PykF) was expressed in a pET system. The full-length of the gene was subcloned into the expression vector pET28a(+) to construct the pET28a-rMGPykF plasmid, which was then transformed into Escherichia coli strain BL21 (DE3) cells. The expression of the 62 kDa recombinant protein of rMGPykF in E. coli strain BL21 (DE3) was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with Coomassie blue staining. Purified rMGPykF exhibited PK catalytic activity, which could reflect the conversion of NADH to NAD(+). Mouse anti-PykF antibodies were generated by immunization of mice with rMGPykF. Immunoblot and immunoelectron microscopy assays identified PykF as an immunogenic protein expressed on the surface of M. gallisepticum cells. Bactericidal assay showed that anti-rMGPykF antiserum killed 70.55% of M. gallisepticum cells, suggesting the protective potential of PykF. Adherence inhibition assay on immortalized chicken fibroblasts (DF-1) cells revealed more than 39.31% inhibition of adhesion in the presence of anti-rMGPykF antiserum, suggesting that PykF of M. gallisepticum participates in bacterial adhesion to DF-1 cells.
Insights
Mycoplasma gallisepticum pyruvate kinase (PykF) is an immunogenic surface protein crucial for bacterial adhesion and survival. Antibodies against PykF demonstrate significant bactericidal activity and inhibit M. gallisepticum adhesion to host cells.
Area of Science:
- Microbiology
- Immunology
- Molecular Biology
Background:
- Mycoplasma gallisepticum causes avian respiratory disease and relies heavily on glycolysis due to incomplete tricarboxylic acid cycle enzymes.
- Pyruvate kinase (PK) is a key glycolytic enzyme, but its immunological properties in Mycoplasma are poorly understood.
Purpose of the Study:
- To characterize the M. gallisepticum pyruvate kinase (PykF) protein.
- To investigate the immunogenicity and potential protective role of PykF against M. gallisepticum infection.
Main Methods:
- Expressed and purified recombinant M. gallisepticum PykF (rMGPykF) in E. coli.
- Generated mouse anti-PykF antibodies.
- Utilized immunoblot, immunoelectron microscopy, bactericidal, and adherence inhibition assays.
Main Results:
- Recombinant PykF exhibited pyruvate kinase activity.
- PykF was identified as an immunogenic surface protein of M. gallisepticum.
- Anti-PykF antibodies showed significant bactericidal activity (70.55%) and inhibited bacterial adhesion to DF-1 cells (39.31%).
Conclusions:
- PykF is an immunogenic surface protein involved in M. gallisepticum adhesion.
- Anti-PykF antibodies possess bactericidal properties and inhibit adhesion, indicating PykF's potential as a vaccine candidate.
