Characterization of pyruvate dehydrogenase complex E1 alpha and beta subunits of Mycoplasma synoviae

Abstract

Mycoplasma synoviae (MS) is an important pathogen, causing enormous economic losses to the poultry industry worldwide every year. Therefore, the studies on MS will lay the foundation for diagnosis, prevention and treatment of MS infection. In this study, primers designed based on the sequences of pyruvate dehydrogenase complex (PDC) E1 alpha and beta subunit genes (pdhA and pdhB, respectively) of MS WVU1853 strain in GenBank were used to amplify the pdhA and pdhB genes of MS WVU1853 strain through PCR. Then the prokaryotic expression vectors pET-pdhA and pET-pdhB were constructed and were expressed in Escherichia coli BL21(DE3) cells. Subsequently, the recombinant proteins rMSPDHA and rMSPDHB were purified and anti-rMSPDHA and anti-rMSPDHB sera were prepared by immunizing rabbits, respectively. Finally, the subcellular localization of PDHA and PDHB in MS, binding activity of rMSPDHA and rMSPDHB to chicken plasminogen (Plg) and human fibronectin (Fn), complement-dependent mycoplasmacidal assays, and adherence and adherence inhibition assays were accomplished. The results showed that PDHA and PDHB were distributed both on the surface membrane and within soluble cytosolic fractions of MS cells. The rMSPDHA and rMSPDHB presented binding activity with chicken Plg and human Fn. The rabbit anti-rMSPDHA and anti-rMSPDHB sera had distinct mycoplasmacidal efficacy in the presence of guinea pig complement, and the adherence of MS to DF-1 cells pretreated with Plg was effectively inhibited by treatment with anti-rMSPDHA or anti-rMSPDHB sera. Hence, the study indicates that the surface-associated MSPDHA and MSPDHB are the adhesion-related factors of MS that contributes to bind to Plg/Fn and adhesion to DF-1 cells.