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

Abstract

Mycoplasma synoviae (MS) is an important pathogen, causing enormous 13 economic losses to the poultry industry worldwide every year. Therefore, the studies on MS 14 will lay the foundation for diagnosis, prevention and treatment of MS infection. In this study, 15 primers designed based on the sequences of pyruvate dehydrogenase complex (PDC) E1 alpha 16 and beta subunit genes (pdhA and pdhB, respectively) of MS WVU1853 strain in GenBank 17 were used to amplify the pdhA and pdhB genes of MS WVU1853 strain through PCR. Then the 18 prokaryotic expression vectors pET-pdhA and pET-pdhB were constructed and were expressed 19 in Escherichia coli BL21(DE3) cells. Subsequently, the recombinant proteins rMSPDHA and 20 rMSPDHB were purified and anti-rMSPDHA and anti-rMSPDHB sera were prepared by 21 immunizing rabbits, respectively. Finally, the subcellular localization of PDHA and PDHB in 22 MS, binding activity of rMSPDHA and rMSPDHB to chicken plasminogen (Plg) and human 23 fibronectin (Fn), complement-dependent mycoplasmacidal assays, and adherence and 24 adherence inhibition assays were accomplished. The results showed that PDHA and PDHB 25 were distributed both on the surface membrane and within soluble cytosolic fractions of MS 26 cells. The rMSPDHA and rMSPDHB presented binding activity with chicken Plg and human 27 Fn. The rabbit anti-rMSPDHA and anti-rMSPDHB sera had distinct mycoplasmacidal efficacy 28 in the presence of guinea pig complement, and the adherence of MS to DF-1 cells pretreated 29 with Plg was effectively inhibited by treatment with anti-rMSPDHA or anti-rMSPDHB sera. 30Hence, the study indicates that the surface-associated MSPDHA and MSPDHB are the 31 adhesion-related factors of MS that contributes to bind to Plg/Fn and adhesion to DF-1 cells. 32 KEYWORDS Mycoplasma synoviae; pyruvate decarboxylase; cloning; biological 33 characteristics 34 Mycoplasma synoviae (MS) is an important pathogen exerting significant economic impact 35 on poultry industry worldwide. MS infections can lead to a range of diseases, from subclinical 36 to severe ailments. In chickens, local infection of MS frequently causes subclinical disease of 37 the upper respiratory tract (Buim et al., 2009; Khiari et al., 2010; Landman, 2014; Wetzel et al., 38 2010), while systemic infection of MS causes synovitis (Wetzel et al., 2010). Furthermore, 39 coinfection of MS with other respiratory pathogens, such as Newcastle disease virus, infectious 40 bursal disease virus, infectious bronchitis virus, Escherichia coli, and Mycoplasma meleagridis, 41can induce airsaculitis, thereby further increasing economic losses (Giambrone et al., 1977; 42 Kleven, 1998;Limpavithayakul et al., 2016;May et al., 2007;Rhoades, 1977; Springer et al., 43 1974;Vardaman et al., 1975). In addition to respiratory tract tropism strains and arthropathic 44 strains, oviduct tropism strains can induce eggshell apex abnormalities without any physical 45 abnormalities (Limpavithayakul et al., 2016). Therefore, research on MS can establish the 46 theoret...