Mycoplasmas are successful pathogens both in humans as well as animals. In cattle, Mycoplasma bovis (M.bovis) is known to be responsible for serious health complications including pneumonia, mastitis and arthritis. However, M. bovis pathogenesis remains unclear. Secreted proteins of M.bovis could influence infection and modify host defense signaling pathways after they enter their extracellular space in the host micro-environment. Therefore this study was aimed to compare the secretomes of M. bovis HB0801 virulent (P1) and attenuated (P150) strain and identify potential pathogenesis-related secreted proteins and biomarkers. The cells of P1 and P150 strain grew in PPLO medium to log-phase and then transferred to PBS for 2 h. Then the supernatant was analyzed by using label-free quantitative proteomics and 477 potential secreted proteins were identified. Combined with the bioinformatics prediction, we found 178 proteins were commonly secreted by P1 and P150 strain, and 49 of them were encoded by mycoplasmal core genes. Additionally, 79 proteins were found to have differential abundance between P1 and P150 strain. Of which proteins, 34 were more abundant and uniquely expressed in P1 indicating a possible association with virulence of M. bovis. Three differentially secreted proteins MbovP0145, MbovP0725, MbovP0174, as well as one equally secreted protein MbovP0481 as positive control and one protein of inner membrane MbovP0310 as negative control were respectively cloned, expressed and evaluated for antigenicity, subcellular locations and the secretion nature with their mouse antisera by western blotting and colony immunoblotting assay. Among them, MbovP0145 was confirmed to be more secreted by P1 than P150 strain, highly reactive with the antisera from naturally infected and P1 experimentally infected cattle but not P150 vaccinated calves indicating its potential as a diagnostic antigen. In conclusion, these findings may represent the most extensive compilation of potentially secreted proteins in mycoplasma species and largest number of differentially secreted proteins between the virulent and attenuated M. bovis strain to date and provide new insights into M. bovis pathogenesis and diagnosis.

中文翻译：

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牛支原体强毒株和减毒株的比较分泌组分析显示MbovP0145是有前途的诊断生物标志物

支原体是人类和动物中成功的病原体。在牛中，牛支原体（M.bovis）引起严重的健康并发症，包括肺炎，乳腺炎和关节炎。但是，牛分枝杆菌的发病机理仍不清楚。牛分枝杆菌的分泌蛋白进入宿主微环境后，可能会影响感染并改变宿主防御信号通路。因此，本研究旨在比较牛分枝杆菌HB0801毒株（P1）和减毒株（P150）的分泌组，并鉴定潜在的病程相关分泌蛋白和生物标记物。P1和P150菌株的细胞在PPLO培养基中生长至对数期，然后转移至PBS中2 h。然后使用无标记定量蛋白质组学分析上清液，并鉴定出477种潜在的分泌蛋白。结合生物信息学预测，我们发现P1和P150菌株通常分泌178种蛋白质，其中49种由支原体核心基因编码。此外，发现79种蛋白质在P1和P150菌株之间具有不同的丰度。其中34个蛋白质含量更高，并且在P1中独特表达，表明可能与牛分枝杆菌的毒力有关。分别克隆，表达并评估了三种差异分泌的蛋白MbovP0145，MbovP0725，MbovP0174以及一种同等分泌的蛋白MbovP0481作为阳性对照，并表达了一种内膜MbovP0310作为阴性对照，Western blotting和菌落免疫印迹法检测小鼠抗血清的亚细胞位置和分泌特性。其中，MbovP0145被证实比P150菌株更能被P1分泌，与天然感染的和经P1实验感染的牛的抗血清高度反应，但未接种P150的牛犊表现出其作为诊断抗原的潜力。总之，这些发现可能代表了支原体物种中潜在分泌蛋白的最广泛汇编，以及迄今为止在有毒和减毒的牛分枝杆菌菌株之间最大数量的差异分泌蛋白，并为牛分枝杆菌的发病机理和诊断提供了新的见识。与天然感染的和经P1实验感染的牛的抗血清具有高度反应性，但未接种P150的牛犊的抗血清具有高度反应性，表明其具有诊断抗原的潜力。总之，这些发现可能代表了支原体物种中潜在分泌蛋白的最广泛汇编，以及迄今为止在有毒和减毒的牛分枝杆菌菌株之间数量最多的差异性分泌蛋白，并为牛分枝杆菌的发病机理和诊断提供了新的见识。与天然感染的和经P1实验感染的牛的抗血清具有高度反应性，但未接种P150的牛犊的抗血清具有高度反应性，表明其具有诊断抗原的潜力。总之，这些发现可能代表了支原体物种中潜在分泌蛋白的最广泛汇编，以及迄今为止在有毒和减毒的牛分枝杆菌菌株之间最大数量的差异分泌蛋白，并为牛分枝杆菌的发病机理和诊断提供了新的见识。