Abstract:[Background] Mycoplasma synoviae (MS) can infect chickens and turkeys to cause airsacculitis, joint exudative, synovial bursitis and tendon sheath synovitis. Previous studies have shown that many metabolism-related enzymes in mycoplasma are present not only in the cytoplasm but also on the cell membrane surface, which act as adhesion associated proteins and play roles on the pathogenicity. The fructose-bisphosphate aldolases (FBAs) have been identified on the membrane and cytoplasmic fractions of Mycoplasma bovis and Mycoplasma gallisepticum, however, the MS FBA has not been studied before. [Objective] To explore the biological functions of metabolism related enzymes in MS, the FBA protein was subjected to bioinformatics analysis, prokaryotic expression, as well as immunogenicity and subcellular localization determinations in this study. [Methods] Using softwares of PSORTb, SignalP 4.1 Server and TMHMM Server to predict the subcellular localization, signal peptides and transmembrane regions of MSFBA. Blastn and MEGA 5.0 were used to analyze the homology and construct the phylogenetic tree of MSFBA. The full length of MS fba gene was amplified by overlap PCR and inserted into the expression vector of pET-28a(+). The recombinant MSFBA (rMSFBA) protein was then expressed in Escherichia coli BL21(DE3) and purified. The immunogenicity of rMSFBA was determined by Western blot analysis using MS-positive chicken serum. The rabbit anti-rMSFBA sera were prepared, and used to perform Western blot against the whole-cell, membrane and cytoplasmic fractions of MS to determine the subcellular localization. Suspension immunofluorescence assay further confirmed the non-membrane surface localization of the MSFBA. [Results] The MSFBA is predicted to be a cytoplasmic protein with no signal peptide and no transmembrane region. It is highly conserved protein which shows up to 99% homology among different MS strains, and 61% to 78% homology with different species of mycoplasma. The phylogenetic tree shows that the MSFBA is evolutionally closed to the FBA from Mycoplasma bovirhinis and Mycoplasma cricetuli, but remote to that of Mycoplasma arginini and Mycoplasma hominis. The rMSFBA protein was successfully expressed and purified, and the relative molecular mass was approximately 33 kD. The rMSFBA presented a specific binding to MS-positive chicken serum, which proved that it had good immunogenicity. Western blot analysis showed the rabbit anti-rMSFBA sera can react with the whole-cell and cytoplasmic fractions of MS bacteria, but not the membrane fraction, indicating the MSFBA protein is distributed in the cytoplasma. Suspension immunofluorescence assay further confirmed the FBA was not displayed on the membrane surface of MS cells. [Conclusion] The MSFBA protein is a highly conserved, immunogenic and cytoplasmic localized protein. The results provide a molecular basis for further study of the biological function of MSFBA protein.
