NADH oxidase of Mycoplasma synoviae is a potential diagnostic antigen, plasminogen/fibronectin binding protein and a putative adhesin

Abstract

Background Mycoplasma synoviae (MS) is an important pathogen causing respiratory diseases and arthritis in chickens and turkeys, thus, resulting in serious economic losses to the poultry industry. Membrane-associated proteins are thought to play important roles in cytoadherence and pathogenesis. NADH oxidase (NOX) is an oxidoreductase involved in glycolysis, which is thought to be a multifunctional protein and potential virulence factor in some pathogens. However, little is known regarding the NOX of MS (MSNOX). We previously demonstrated that MSNOX was a metabolic enzyme distributed in not only the cytoplasm but also the MS membrane. This study was aimed at exploring NOX’s potential as a diagnostic antigen and its role in MS cytoadherence. Results Western blots and ELISAs indicated that recombinant MSNOX (rMSNOX) protein reacted with sera positive for various MS isolates, but not MG isolates or other avian pathogens, thus, suggesting that rMSNOX is a potential diagnostic antigen. In addition, rabbit anti-rMSNOX serum showed substantial complement-dependent mycoplasmacidal activity toward various MS isolates and MG Rlow. MSNOX protein was found not only in the cytoplasm but also on the membrane of MS through suspension immunofluorescence and immunogold electron microscopy assays. Indirect immunofluorescence assays indicated that rMSNOX adhered to DF-1 cells, and this adherence was inhibited by rabbit anti-rMSNOX, but not anti-MG serum. Furthermore, indirect immunofluorescence and colony counting assays confirmed that the rabbit anti-rMSNOX serum inhibited the adherence of various MS isolates but not MG Rlow to DF-1 cells. Moreover, plasminogen (Plg)- and fibronectin (Fn)-binding assays demonstrated that rMSNOX bound Plg and Fn in a dose-dependent manner, thereby further confirming that MSNOX may be a putative adhesin. Conclusion MSNOX was identified to be a surface immunogenic protein that has good immunoreactivity and specificity in Western blot and ELISA, and therefore, may be used as a potential diagnostic antigen in the future. In addition, rMSNOX adhered to DF-1 cells, an effect inhibited by rabbit anti-rMSNOX, but not anti-MG serum, and anti-rMSNOX serum inhibited the adherence of various MS isolates, but not MG Rlow, to DF-1 cells, thus indicating that the inhibition of adherence by anti-MSNOX serum was MS specific. Moreover, rMSNOX adhered to extracellular matrix proteins including Plg and Fn, thus suggesting that NOX may play important roles in MS cytoadherence and pathogenesis. Besides, rabbit anti-rMSNOX serum presented complement-dependent mycoplasmacidal activity toward both MS and MG, indicating the MSNOX may be further studied as a potential protective vaccine candidate.

Discrimination of mycoplasma infection using machine learning models trained on autofluorescence signatures of host cells

“…Mycoplasma NADH oxidase is reported to be a potential mediator of virulence, [22][23][24] implying that mycoplasma infection causes an increase in the NADH oxidase activity in host cells. NADH is a major cellular autofluorescence molecule with excitation and emission peaks of 330-360 nm and 440-470 nm, respectively.…”

Mycoplasma synoviae elongation factor thermo stable is an adhesion-associated protein that enters cells by endocytosis and stimulates DF-1 cell proliferation.

“…Membrane proteins were extracted from DF-1 cells using a Membrane Protein Extraction Kit (Solarbio, Beijing, China). ELISA was performed as previously described [14]: 500 ng of cell membrane proteins were encapsulated in each well of the ELISA plate, which was blocked using 5% skim milk. rEF-Ts and anti-rEF-Ts serum pretreated with rEF-Ts were added to the wells.…”

“…Despite the screening of several M. synoviae immunogenic adhesion-associated proteins in recent years, including α-Eno [13], NADH [14], DLD [15], PDHA [16], PDHB [16], P35 [17], P78 [18], Vlha [19], and EF-Tu [19], signi cant challenges persist in designing an effective M. synoviae vaccine. Membrane proteins are key Mycoplasma proteins and are integral to various microbial activities.…”

Novel antigenic proteins of Mycoplasma synoviae as potential vaccine candidates

“…To detect the adherence of rRS01790, rBMP, rGrpE, rRS00900 and rRS00275 to DF-1 cells, indirect immunofluorescence assay was used as described previously with some modifications 11 . After incubation, cells were washed three times with PBS, and then labeled with mouse anti-His-tag IgG (1:1000) and anti-rabbit IgG-FITC (1:1000; Sigma) for 1 h respectively.…”

“…Subunit vaccine has been extremely difficult to acquire due to the lack of understanding of M. synoviae proteins. Only a few of M. synoviae antigens have been reported, such as enolase 8 , pyruvate dehydrogenase complex E1 alpha, beta subunits (PDHA and PDHB), dihydrolipoamide dehydrogenase (PdhD) 9 , P35 10 and NADH oxidase (NOX) 11 . These studies have promoted the understanding of the biological function and the role in the pathogenesis of M. synoviae in proteins.…”