Manjarrez-Hernandez A, Gavilanes-Parra S, Chavez-Berrocal M E, Molina-Lopez J, Cravioto A
Department of Public Health, Faculty of Medicine, National Autonomous University, Mexico City, Mexico.
Infect Immun. 1997 Nov;65(11):4494-501. doi: 10.1128/iai.65.11.4494-4501.1997.
We have detected human intestinal brush border proteins to which Escherichia coli strains adhere by means of a blotting-nitrocellulose method in which the binding of radiolabeled bacteria to sodium dodecyl sulfate-polyacrylamide gel electrophoresis-separated intestinal cell membranes was evaluated. The brush border fraction contained several polypeptides that bound only adherent E. coli strains. The most prominent and consistent of these proteins had apparent molecular masses of 32 to 33 kDa. Additional polypeptides ranging from 50 to 70, from 105 to 130, and from 180 to 200 kDa were also recognized by adherent E. coli strains, although with less intensity (in accordance with the number of bound bacteria to these polypeptides). Independently of the pattern of adherence (localized [LA], diffuse [DA], or aggregative [AggA]) all HEp-2-adhering strains recognized, with different intensities, the 32- to 33-kDa brush border proteins, whereas nonadhesive strains did not. The relative avidity of an LA strain to bind to the 32- to 33-kDa proteins was approximately seven- and sixfold higher than the binding of strains with aggregative and diffuse adherence, respectively. Thus, it is reasonable to think that LA, DA, and AggA strains have a common adhesin that mediates binding to the 32- to 33-kDa bands. Inhibition experiments using HEp-2 cells demonstrated that isolated 32- to 33-kDa proteins or specific antiserum blocked preferentially bacterial adherence of the LA pattern. Delipidization and protein digestion of the human brush borders confirmed that E. coli bound to structures of a proteinaceous nature. Deglycosylation studies and sodium meta-periodate oxidation of the intestinal cell membranes decreased bacterial binding activity significantly, indicating that E. coli bound to carbohydrate moieties in the glycoproteins. These results suggest that binding of E. coli strains, mainly of the LA phenotype, to the 32- to 33-kDa proteins could play a role in colonization through adherence to the intestinal mucosa.
我们通过一种印迹-硝酸纤维素方法检测到了大肠杆菌菌株所黏附的人肠刷状缘蛋白，该方法用于评估放射性标记细菌与十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分离的肠细胞膜的结合情况。刷状缘部分含有几种仅与黏附性大肠杆菌菌株结合的多肽。其中最突出且一致的蛋白质表观分子量为32至33 kDa。黏附性大肠杆菌菌株还识别出了分子量在50至70 kDa、105至130 kDa以及180至200 kDa范围内的其他多肽，不过结合强度较低（与这些多肽结合的细菌数量一致）。无论黏附模式如何（局部黏附[LA]、弥漫性黏附[DA]或聚集性黏附[AggA]），所有黏附于HEp-2细胞的菌株都能以不同强度识别32至33 kDa的刷状缘蛋白，而非黏附性菌株则不能。一株LA菌株与32至33 kDa蛋白质结合的相对亲和力分别比具有聚集性和弥漫性黏附的菌株高约7倍和6倍。因此，有理由认为LA、DA和AggA菌株具有一种共同的黏附素，介导与32至33 kDa条带的结合。使用HEp-2细胞的抑制实验表明，分离出的32至33 kDa蛋白质或特异性抗血清优先阻断了LA模式的细菌黏附。人刷状缘的脱脂和蛋白质消化证实大肠杆菌与蛋白质性质的结构结合。肠细胞膜的去糖基化研究和高碘酸钠氧化显著降低了细菌结合活性，表明大肠杆菌与糖蛋白中的碳水化合物部分结合。这些结果表明，大肠杆菌菌株，主要是LA表型的菌株，与32至33 kDa蛋白质的结合可能通过黏附于肠黏膜在定植过程中发挥作用。
