Identification of Secreted O-Mannosylated Proteins From BCG and Characterization of Immunodominant Antigens BCG_0470 and BCG_0980
Identification of Secreted O-Mannosylated Proteins From BCG and Characterization of Immunodominant Antigens BCG_0470 and BCG_0980
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BCG 分泌的 O-甘露糖化蛋白的鉴定以及免疫显性抗原 BCG_0470 和 BCG_0980 的表征
DOI：
10.3389/fmicb.2020.00407
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发表时间：
2020-03-13
影响因子：
5.2
通讯作者：
Yang, Shufeng
中科院分区：
文献类型：
--
作者：
Deng, Guoying;Zhang, Wenli;Yang, Shufeng
细菌糖蛋白已被研究作为候选疫苗以及诊断生物标志物。然而，它们在牛分枝杆菌菌株卡介苗（BCG），结核分枝杆菌的非致病性模型中知之甚少。为了了解BCG分泌的O-甘露糖基化糖蛋白的作用，我们进行了ConA凝集素亲和层析和质谱分析，以确定O-甘露糖基化蛋白在BCG培养滤液。随后使用从BCG免疫小鼠获得的多克隆抗体进行抗原筛选，最终鉴定出15种内源性O-甘露糖基化蛋白。其中，BCG_0470和BCG_0980（PstS 3）被揭示为免疫显性抗原。为了检测抗原的保护作用，首先在耻垢分枝杆菌和大肠杆菌中表达重组抗原蛋白，然后将纯化的蛋白用于加强BCG致敏小鼠。总体而言，治疗的小鼠在体内表现出更大的迟发型超敏反应，以及更强的Th 1反应，包括更高水平的IFN-γ，TNF-α和特异性IgG。因此，甘露糖基化蛋白BCG_0470和BCG_0980有效地放大了BCG在小鼠中诱导的免疫应答。总之，我们的研究结果表明，含有甘露糖的寡糖链是糖蛋白的抗原决定簇，为未来的疫苗优化和设计提供了关键的见解。
Bacterial glycoproteins have been investigated as vaccine candidates as well as diagnostic biomarkers. However, they are poorly understood in Mycobacterium bovis strain bacille Calmette-Guerin (BCG), a non-pathogenic model of Mycobacterium tuberculosis. To understand the roles of secreted O-mannosylated glycoproteins in BCG, we conducted a ConA lectin-affinity chromatography and mass spectra analysis to identify O-mannosylated proteins in BCG culture filtrate. Subsequent screening of antigens was performed using polyclonal antibodies obtained from a BCG-immunized mouse, with 15 endogenous O-mannosylated proteins eventually identified. Of these, BCG_0470 and BCG_0980 (PstS3) were revealed as the immunodominant antigens. To examine the protective effects of the antigens, recombinant antigens proteins were first expressed in Mycobacterium smegmatis and Escherichia coli, with the purified proteins then used to boost BCG primed-mice. Overall, the treated mice showed a greater delayed-type hypersensitivity response in vivo, as well as stronger Th1 responses, including higher level of IFN-gamma, TNF-alpha, and specific-IgG. Therefore, mannosylated proteins BCG_0470 and BCG_0980 effectively amplified the immune responses induced by BCG in mice. Together, our results suggest that the oligosaccharide chains containing mannose are the antigenic determinants of glycoproteins, providing key insight for future vaccine optimization and design.