Aragón V, Díaz R, Moreno E, Moriyón I
Departamento de Microbiología, Universidad de Navarra, Pamplona, Spain.
J Bacteriol. 1996 Feb;178(4):1070-9. doi: 10.1128/jb.178.4.1070-1079.1996.
Brucella native haptens (NHs) extracted with hot water from smooth (S)-type B. abortus and B. melitensis were purified to high levels of serological activity and compared with the polysaccharide obtained by acid hydrolysis (PS) of the S lipopolysaccharide (S-LPS). By 13C nuclear magnetic resonance analysis, NHs showed the spectrum of a homopolymer of alpha-1,2- or alpha-1,2- plus alpha-1,3-linked 4-formamido-4,6-dideoxy-D-mannose (N-formylperosamine) previously reported for the LPS O chain. However, while PS contained up to 0.6% 3-deoxy-D-manno-2-octulosonate, this LPS-core marker was absent from NH. High performance liquid chromatography and thin-layer chromatography showed heterogeneity in NH purified from whole cells but not in PS. By immunoprecipitation, polysaccharides indistinguishable from NH were demonstrated in extracts obtained with phenol-water, saline at 60 degrees C, and ether-water treatments, and none of these treatments caused S-LPS hydrolysis detectable with antibodies to the O chain and lipid A. Two lines of evidence showed that NH was in the cell surface. First, NH became biotinylated when B. abortus live cells were labelled with biotin-hydrazide, and the examination of cell fractions and electron microscopy sections with streptavidin-peroxidase and streptavidin-coloidal gold, respectively, showed that labelling was extrinsic. Moreover, whereas only traces of NH were found in cytosols, the amount of NH was enriched in cell envelopes and in the outer membrane blebs spontaneously released by brucellae during growth. Interactions between NH and S-LPS were observed in crude cell extracts, and such interactions could be reconstituted by using purified NH and LPS. The results demonstrate that NH is not a hydrolytic product of S-LPS and suggest a model in which LPS-independent O-type polysaccharides (NH) are intertwined with the O chain in the outer membrane of S-type brucellae.
用热水从光滑型流产布鲁氏菌和羊布鲁氏菌中提取的布鲁氏菌天然半抗原（NHs）被纯化至具有高血清学活性水平，并与S型脂多糖（S-LPS）经酸水解得到的多糖（PS）进行比较。通过13C核磁共振分析，NHs显示出先前报道的LPS O链的α-1,2-或α-1,2-加α-1,3-连接的4-甲酰胺基-4,6-二脱氧-D-甘露糖（N-甲酰基过氧胺）同聚物的光谱。然而，虽然PS含有高达0.6%的3-脱氧-D-甘露糖-2-辛酮酸，但该LPS核心标记物在NH中不存在。高效液相色谱和薄层色谱显示，从全细胞中纯化的NH存在异质性，而PS中不存在。通过免疫沉淀，在用酚-水、60℃生理盐水和醚-水处理获得的提取物中证明了与NH无法区分的多糖，并且这些处理均未导致用O链和脂质A抗体可检测到的S-LPS水解。有两条证据表明NH位于细胞表面。首先，当用生物素酰肼标记流产布鲁氏菌活细胞时，NH会被生物素化，分别用抗生物素蛋白-过氧化物酶和抗生物素蛋白-胶体金检查细胞组分和电子显微镜切片表明标记是外在的。此外，虽然在细胞溶质中仅发现痕量的NH，但NH的量在细胞包膜和布鲁氏菌生长过程中自发释放的外膜泡中富集。在粗细胞提取物中观察到NH与S-LPS之间的相互作用，并且使用纯化的NH和LPS可以重建这种相互作用。结果表明NH不是S-LPS的水解产物，并提出了一个模型，其中不依赖LPS的O型多糖（NH）与光滑型布鲁氏菌外膜中的O链相互缠绕。
