Characterization of Brucella abortus and Brucella melitensis native haptens as outer membrane O-type polysaccharides independent from the smooth lipopolysaccharide
Characterization of Brucella abortus and Brucella melitensis native haptens as outer membrane O-type polysaccharides independent from the smooth lipopolysaccharide
复制标题
DOI：
10.1128/jb.178.4.1070-1079.1996
复制
发表时间：
1996-02-01
影响因子：
3.2
通讯作者：
Moriyon, I
中科院分区：
文献类型：
--
作者：
Aragon, V;Diaz, R;Moriyon, I
用热水提取的S型流产布鲁氏菌和羊种布鲁氏菌半抗原具有较高的血清学活性，并与S脂多糖酸水解法得到的多糖(S-脂多糖)进行比较。通过C-13核磁共振分析，NHS显示了一种由α-1，2-或α-1-1，2-加上α-1，3-连接的4-甲酰胺-4，6-二脱氧-D-甘露糖(N-甲酰基过氧甘露糖)的均聚物(N-甲酰基过氧甘露糖)的光谱。然而，当PS中含有高达0.6%的3-脱氧-D-甘露-2-辛磺酸钠时，NH中不存在这个内毒素核心标志。高效液相色谱和薄层层析在全细胞纯化的NH中表现出异质性，而在PS中则不是。通过免疫沉淀，在苯酚-水、60℃生理盐水和乙醚-水处理的提取物中发现了与NH难以区分的多糖，这些处理都没有引起O链抗体和脂质A抗体检测到的S-内毒素的水解。两条证据表明NH存在于细胞表面。首先，当流产杆菌活细胞被生物素-酰肼标记时，NH被生物素标记，分别用链霉亲和素-过氧化物酶和链霉亲和素-胶体金标记细胞组分和电子显微镜切片，表明标记是外在的。此外，在胞质中只发现了微量的NH，而在细胞被膜和布鲁氏菌在生长过程中自发释放的外膜气泡中则有大量的NH。在细胞粗提液中观察到NH与S-内毒素的相互作用，这种相互作用可以用纯化的NH和内毒素重建。结果表明，NH不是S-内毒素的水解物，并提出了S布鲁氏菌外膜O链与非内毒素非依赖性O-型多糖缠绕的模型。
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 C-13 nuclear magnetic resonance analysis, NHs showed the spectrum of a homopolymer of alpha-1,2- or alpha-1-1,2- 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.