Abstract
Mycoplasma gallisepticum is a causative agent of chronic respiratory disease in chickens, typically causing great economic losses. Cytoadherence is the critical stage for mycoplasma infection, and the associated proteins are important for mycoplasma pathogenesis. Many glycolytic enzymes are localized on the cell surface and can bind the extracellular matrix of host cells. In this study, the M. gallisepticum pyruvate dehydrogenase E1 alpha subunit (PDHA) and beta subunit (PDHB) were expressed in Escherichia coli, and their enzymatic activities were identified based on 2,6-dichlorophenol indophenol reduction. When recombinant PDHA (rPDHA) and recombinant PDHB (rPDHB) were mixed at a 1:1 molar ratio, they exhibited strong enzymatic activity. Alone, rPDHA and rPDHB exhibited no or weak enzymatic activity. Further experiments indicated that both PDHA and PDHB were surface-exposed immunogenic proteins of M. gallisepticum. Bactericidal assays showed that the mouse anti-rPDHA and anti-rPDHB sera killed 48.0% and 75.1% of mycoplasmas respectively. A combination of rPDHA and rPDHB antisera had a mean bactericidal rate of 65.2%, indicating that rPDHA and rPDHB were protective antigens, and combining the two sera did not interfere with bactericidal activity. Indirect immunofluorescence and surface display assays showed that both PDHA and PDHB adhered to DF-1 chicken embryo fibroblast cells and adherence was significantly inhibited by antisera against PDHA and PDHB. Adherence inhibition of M. gallisepticum to DF-1 chicken embryo fibroblast cells was 30.2% for mouse anti-rPDHA serum, 45.1% for mouse anti-rPDHB serum and 72.5% for a combination of rPDHA and rPDHB antisera, suggesting that rPDHA and rPDHB antisera may have synergistically interfered with M. gallisepticum cytoadherence. Plasminogen (Plg)-binding assays further demonstrated that both PDHA and PDHB were Plg-binding proteins, which may have contributed to bacterial colonization. Our results clarified the enzymatic activity of M. gallisepticum PDHA and PDHB and demonstrated these compounds as Plg-binding proteins involved in cytoadherence.
Highlights
Mycoplasma gallisepticum is one of the most important avian pathogens
Full-length pdhA (1080 bp) and pdhB (978 bp) gene fragments were obtained from overlapping PCR amplification (Fig 1A), cloned into pET-28a (+) and transformed into E. coli BL21
The enzymatic activities of recombinant PDHA (rPDHA) and recombinant PDH E1 beta subunit (PDHB) (rPDHB) were measured by detecting reduction of 2,6-DCPIP at OD600
Summary
Mycoplasma gallisepticum is one of the most important avian pathogens. It is the primary agent of chronic respiratory disease in chickens and infectious sinusitis in turkeys, causing great economic losses in the poultry industry worldwide [1]. Studies revealed that some glycolytic enzymes, including glyceraldehyde-3-phosphate dehydrogenase (GAPDH) [11,12,13,14] and α-enolase (Eno) [15,16,17,18], are on the surface of mycoplasmas. They are involved in cytoadherence through interactions with host components such as plasminogen (Plg) [17,18,19,20], fibronectin (Fn) [11, 18], mucin [12] and β-actin [14, 16]. Further investigation of moonlighting proteins is helpful for enrichment of the adhesion-related proteins in M. gallisepticum and for better understanding the organization of the parasitic lifestyle of mycoplasmas
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