Doig P, Trust T J
Department of Biochemistry and Microbiology, University of Victoria, British Columbia, Canada.
Infect Immun. 1994 Oct;62(10):4526-33. doi: 10.1128/iai.62.10.4526-4533.1994.
Despite the potential significance of surface-localized antigens in the colonization by and disease processes of Helicobacter pylori, few such components have been unequivocally identified and/or characterized. To further investigate the surface of this bacterium, monoclonal antibodies (MAbs) to a sarcosine-insoluble outer membrane fraction prepared from H. pylori NCTC 11637 were raised. MAbs were selected on the basis of their surface reactivity to whole cells by enzyme-linked immunosorbent assay, immunofluorescence, and immunoelectron microscopy. By use of this selection protocol, 14 surface-reactive MAbs were chosen. These MAbs were used to identify six protein antigens (molecular masses, 80, 60, 51, 50, 48, and 31 kDa), all of which were localized within or associated with the outer membrane. Two of the MAbs recognized the core region of lipopolysaccharide (LPS). Only these two anti-LPS MAbs also recognized the flagellar sheath, indicating a structural difference between the sheath and outer membrane. Three of the protein antigens (80, 60, and 51 kDa) were strain specific, while the other three antigens were present in other strains of H. pylori. Both the 51- and 48-kDa antigens were heat modifiable and likely are porins. A conserved 31-kDa protein may represent another species of porin. A method involving sucrose density ultracentrifugation and Triton extraction that allows the preparation of H. pylori outer membranes with minimal inner membrane contamination is described. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis showed that the protein content of the H. pylori outer membrane is similar structurally to those of other species of Helicobacter but markedly different from those of taxonomically related Campylobacter spp. and Escherichia coli. H. pylori also appeared to lack peptidoglycan-associated proteins.
尽管表面定位抗原在幽门螺杆菌的定植和疾病过程中具有潜在重要性，但明确鉴定和/或表征的此类成分却很少。为了进一步研究这种细菌的表面，制备了针对从幽门螺杆菌NCTC 11637分离的肌氨酸不溶性外膜组分的单克隆抗体（MAb）。通过酶联免疫吸附测定、免疫荧光和免疫电子显微镜，根据单克隆抗体对全细胞的表面反应性进行筛选。采用这种筛选方案，挑选出14种表面反应性单克隆抗体。这些单克隆抗体用于鉴定6种蛋白质抗原（分子量分别为80、60、51、50、48和31 kDa），所有这些抗原都定位于外膜内或与外膜相关。其中两种单克隆抗体识别脂多糖（LPS）的核心区域。只有这两种抗LPS单克隆抗体也识别鞭毛鞘，表明鞘与外膜之间存在结构差异。三种蛋白质抗原（80、60和51 kDa）具有菌株特异性，而其他三种抗原存在于幽门螺杆菌的其他菌株中。51 kDa和48 kDa抗原均可被热修饰，可能是孔蛋白。一种保守的31 kDa蛋白可能代表另一种孔蛋白。描述了一种涉及蔗糖密度超速离心和Triton提取的方法，该方法可制备内膜污染最小的幽门螺杆菌外膜。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分析表明，幽门螺杆菌外膜的蛋白质含量在结构上与其他幽门螺杆菌物种相似，但与分类学相关的弯曲杆菌属物种和大肠杆菌明显不同。幽门螺杆菌似乎也缺乏肽聚糖相关蛋白。
