Biological Characteristics Of The Two Surface-Localised Glycolytic Enzymes In Mycoplasma Gallisepticum

The widespread avian pathogen Mycoplasma gallisepticum is a causative agent of chronic respiratory disease in chicken and infectious sinusitis in tuikeys. As these wall-less prokaryotes lack some tricarboxylic acid cycle enzymes, the glycolysis metabolic pathway is of great importance to these organisms. Adolase(FBA) and Pyruvate kinase (pykF) are two of the key enzymes of the glycolytic pathway, and their immunological characteristics in mycoplasmas are not well known.1. Biological functions of adolase in M.gallisepticumAccording to published gene sequence of M. gallisepticum, sequence specific primers were designed and used for Overlap PCR amplifation of fba gene from M.gallisepticum strain Rlow. The PCR product was cloned into expression vector pET28a(+) and subsequently sequenced. The result showed that the full-length offba is873bp in length, encoding290amino acids. The constructed recombinant plasmid pET28a-fba was then introduced into Escherichia coli strain BL21(DE3) competent cells for IPTG-inducible expression of rMGfba. Purified rMGfba exhibited aldolase catalytic activity indicating that it was correctly expressed in vitro. Mouse antiserum to fba was generated by immunization with rMGfba. Western-blot assay and transmission electron microscope(TEM)with the antiserum identified FBA be presented on the surface of M. gallisepticum cells. Adherence inhibition assay on immortalized chicken fibroblasts (DF-1) cells revealed more than42.68%inhibition of adhesion in the presence of anti-rMGfba antiserum, suggesting that FBA of M. gallisepticum participates in bacterial adhesion to DF-1cells. These results lay the foundation for the further research of FBA.2. Biological functions of pyruvate kinase in M.gallisepticumIn this study, the M. gallisepticum pyruvate kinase(pykF) fusion protein rMGpykF was expressed in a pET system. The full-length gene was subcloned into the expression vector pET28a(+) to construct the pET-pykF plasmid, and transformed into E. coli strain DE3cells. The expression of the60kDa rMGpykF in E. coli 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 assay identified pykF as an immunogenic protein expressed on the surface of M. gallisepticum cells. Bactericidal assay showed that anti-rMGpykF antiserum killed66.16%of M. gallisepticum cells, suggesting the protective potential of pykF. Adherence inhibition assay on immortalized chicken fibroblasts (DF-1) cells revealed more than39.13%inhibition of adhesion in the presence of anti-rMGpykF antiserum, suggesting that pykF of M. gallisepticum participates in bacterial adhesion to DF-1cells.