背景:滑膜支原体(MS)是引起鸡和火鸡呼吸道疾病和关节炎的重要病原体,因此,给家禽业造成了严重的经济损失。膜相关蛋白被认为在细胞粘附和发病机理中起重要作用。NADH氧化酶(NOX)是一种参与糖酵解的氧化还原酶,它被认为是一种多功能蛋白和一些病原体的潜在毒力因子。然而,关于MS的NOX(MSNOX)知之甚少。我们先前证明MSNOX是一种代谢酶,不仅分布在细胞质中,而且分布在MS膜中。本研究旨在探讨NOX作为诊断抗原的潜力及其在MS细胞粘附中的作用。
结果:Western印迹和ELISA表明,重组MSNOX(rMSNOX)蛋白与各种MS分离株的血清呈阳性反应,但不是MG分离株或其他禽类病原体,因此,表明rMSNOX是一种潜在的诊断抗原。此外,兔抗rMSNOX血清对各种MS分离株和MGRlow显示出大量补体依赖性支原体活性。通过悬浮免疫荧光和免疫金电子显微镜测定,不仅在MS的细胞质中,而且在MS的膜上也发现了MSNOX蛋白。间接免疫荧光分析表明rMSNOX粘附于DF-1细胞,这种粘附被兔抗rMSNOX抑制,但不是抗MG血清.此外,间接免疫荧光和菌落计数测定证实,兔抗rMSNOX血清抑制各种MS分离株的粘附,但不抑制MGRlow对DF-1细胞的粘附。此外,纤溶酶原(Plg)和纤连蛋白(Fn)结合试验证明rMSNOX以剂量依赖性方式结合Plg和Fn,从而进一步证实MSNOX可能是推定的粘附素。
结论:MSNOX被鉴定为一种表面免疫原性蛋白,在Westernblot和ELISA中具有良好的免疫反应性和特异性,因此,有可能作为一种潜在的诊断抗原。此外,rMSNOX粘附于DF-1细胞,兔抗rMSNOX抑制的效果,但不是抗MG血清,抗rMSNOX血清抑制各种MS分离株的粘附,但不是MGRlow,到DF-1细胞,因此表明抗MSNOX血清对粘附性的抑制是MS特异性的。此外,rMSNOX粘附于细胞外基质蛋白,包括Plg和Fn,因此表明NOX可能在MS细胞粘附和发病机制中起重要作用。此外,兔抗rMSNOX血清对MS和MG均具有补体依赖性支原体活性,表明MSNOX可以作为潜在的保护性疫苗候选物进行进一步研究。
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.
CONCLUSIONS: 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.