Wellcome-Wolfson Institute of Experimental Medicine, Queen's University Belfast , Belfast, UK.
Department of Microbiology and Immunology, University of Melbourne at the Peter Doherty Institute for Infection and Immunity , Melbourne, Australia.
Virulence. 2021 Dec;12(1):493-506. doi: 10.1080/21505594.2021.1876440.
We recently described a protein -glycosylation pathway conserved in all species of the genus that results in the synthesis and incorporation of a trisaccharide glycan to membrane-exported proteins. Here, we exploited this system to construct and evaluate a diagnostic tool for glanders. , the causative agent of glanders, is a highly infectious and fatal zoonotic pathogen that infects horses, mules, donkeys, and occasionally humans. A highly sensitive and specific diagnostic tool is crucial for the control, elimination, and eradication of infections. We constructed plasmids carrying synthetic genes encoding a modified, previously unannotated glycoprotein containing three glycosylation sequons fused to the cholera toxin B-subunit. The resulting proteins were glycosylated in the K56-2 parental strain, but not in glycosylation-deficient mutants, as determined by SDS-PAGE and fluorescent lectin blots. One of these glycoproteins was used as an antigen in ELISA and western blots to screen a panel of serum samples collected from glanders-infected and healthy horses, which were previously investigated by complement fixation test and indirect ELISA based on a semi-purified fraction of . We show that ELISA and western blot assays based on our glycoprotein antigen provide 100% specificity, with a sensitivity greater than 88%. The glycoprotein antigen was recognized by serum samples collected from patients infected with and . Our results indicate that protein -glycosylation in can be exploited as a biomarker for diagnosis of -associated infections.
我们最近描述了一个在该属的所有物种中保守的蛋白质糖基化途径，该途径导致三糖聚糖的合成和掺入到膜输出蛋白中。在这里，我们利用这个系统构建并评估了一种用于鼻疽的诊断工具。鼻疽伯克霍尔德菌是一种高度传染性和致命的人畜共患病病原体，感染马、骡、驴，偶尔也感染人类。一种高度敏感和特异性的诊断工具对于控制、消除和根除感染至关重要。我们构建了携带合成基因的质粒，这些基因编码一种经过修饰的、以前未注释的糖蛋白，该蛋白含有融合到霍乱毒素 B 亚单位的三个糖基化序列。通过 SDS-PAGE 和荧光凝集素印迹法测定，在 K56-2 亲本菌株中这些蛋白发生了糖基化，但在糖基化缺陷突变体中没有发生，在糖基化缺陷突变体中没有发生。其中一种糖蛋白被用作 ELISA 和 Western blot 的抗原，以筛选一组来自鼻疽感染和健康马的血清样本，这些样本先前通过补体结合试验和基于半纯化 的间接 ELISA 进行了研究。我们表明，基于我们的糖蛋白抗原的 ELISA 和 Western blot 检测方法具有 100%的特异性，敏感性大于 88%。该糖蛋白抗原被来自感染 和 的患者的血清样本识别。我们的结果表明，鼻疽中的蛋白质糖基化可以被用作与感染相关的诊断标志物。
