A secreted protein of Mycoplasmopsis synoviae, SSB, is a critical adhesion factor that promotes the enrichment of Annexin A2 at the cell membrane.

Mycoplasmopsis synoviae is an important pathogen in poultry that primarily causes synovitis, tenosynovitis, eggshell abnormalities, and respiratory diseases, resulting in significant economic losses to the poultry industry worldwide. The aim of this study was to systematically screen and characterize the proteins secreted by M. synoviae and to investigate the role of SSB in M. synoviae infection. We first identified 13 proteins secreted by M. synoviae using liquid chromatography‒tandem mass spectrometry analysis. Bioinformatic analysis of SSBs revealed that they lack a transmembrane domain and the characteristic features of a cleavable signal peptide and are potential virulence factors. After induced expression and purification of SSB from Escherichia coli, rabbit anti-SSB serum was prepared. Subcellular localization analysis by immunogold labeling and Western blotting further confirmed that SSB is present mainly in the cytoplasm and culture supernatant of M. synoviae. ELISA and indirect immunofluorescence analyses revealed that SSB binds to membrane proteins and adheres to the surface of chicken DF-1 cells and that rabbit anti-SSB serum significantly inhibits M. synoviae adhesion to DF-1 cells. In vitro experiments confirmed that SSB could interact directly with plasminogen and fibronectin in a dose-dependent manner. In addition, our study revealed that SSB significantly promoted Annexin A2 (AnxA2) protein expression in DF-1 cells during adhesion, significantly colocalized with AnxA2, and promoted its enrichment at the membrane. These findings offer a novel experimental foundation for understanding the molecular mechanisms underlying the role of the secreted protein SSB in M. synoviae infection.IMPORTANCEPathogenic bacteria transport proteins to the extracellular compartment through a variety of secretion systems, and most of these secreted proteins act as adhesins, toxins, or immunomodulatory molecules in host cells, playing an important role in bacterial pathogenesis. However, studies on secreted proteins of Mycoplasmopsis synoviae are still incomplete. In our study, we systematically screened and identified the secreted proteins of M. synoviae strain WVU1853 for the first time and conducted an in-depth investigation of the role of SSB, one of the secreted proteins, in the infection process, in vitro. Our results not only reveal the key role of SSB in the infection process of M. synoviae but also provide a new theoretical basis for studying the invasion mechanisms of Mycoplasma-secreted proteins and related processes.