Postnova T, Gómez-Duarte O G, Richardson K
Biosciences Division, General Atomics, San Diego, CA 92186-9784, USA.
Microbiology (Reading). 1996 Oct;142 ( Pt 10):2767-76. doi: 10.1099/13500872-142-10-2767.
Vibrio cholerae must colonize the human small intestine to cause diarrhoeal disease. V.cholerae strains N16961 (EI Tor, Inaba) and 395 (classical, Ogawa) adhered to the epithelial cell surface and the mucus layer of isolated human small intestinal epithelial cells. They adhered specifically to the mucosa and apical membrane in thin sections of small intestine. No binding to the basolateral membrane of dissected epithelial tissue or to intracellular components of the epithelial cells was observed by either light or indirect immunofluorescence microscopy. Based on these results, a modified ELISA was developed to quantitatively study adherence of V. cholerae to human small intestinal epithelial cells. The assay used homogenized human small intestinal mucosal tissue as the substrate for binding. Treatment of the epithelial cell homogenate with 2-mercaptoethanol to disrupt protein and glycoprotein secondary structure inhibited the binding of V. cholerae strains, suggesting that binding was to specific receptors. Several V. cholerae strains and mutants from both biotypes were tested for adherence in the modified ELISA. Wild-type strains of both biotypes and non-enterotoxigenic strains, which were known to colonize humans, adhered. V. cholerae mutants defective in motility, flagellar structure of chemotaxis, which were known to exhibit reduced colonization in animal models, exhibited decreased adherence. The specificity of the assay and its ability to quantify binding should facilitate identification and the study of adherence factors involved in the colonization of human small intestinal epithelial cells by V. cholerae.
霍乱弧菌必须在人类小肠中定殖才能引发腹泻病。霍乱弧菌菌株N16961（埃尔托生物型，稻叶型）和395（古典生物型，小川型）黏附于分离出的人小肠上皮细胞的上皮细胞表面和黏液层。它们特异性地黏附于小肠薄片中的黏膜和顶端膜。通过光学显微镜或间接免疫荧光显微镜观察，未发现其与解剖的上皮组织的基底外侧膜或上皮细胞的细胞内成分有结合。基于这些结果，开发了一种改良的酶联免疫吸附测定法（ELISA）来定量研究霍乱弧菌对人小肠上皮细胞的黏附。该测定法使用匀浆的人小肠黏膜组织作为结合底物。用2-巯基乙醇处理上皮细胞匀浆以破坏蛋白质和糖蛋白的二级结构，抑制了霍乱弧菌菌株的结合，这表明结合是针对特定受体的。对来自两种生物型的几种霍乱弧菌菌株和突变体进行了改良ELISA中的黏附测试。两种生物型的野生型菌株以及已知能在人类中定殖的非产肠毒素菌株都有黏附。已知在动物模型中定殖能力降低的运动性、鞭毛结构或趋化性有缺陷的霍乱弧菌突变体，其黏附能力下降。该测定法的特异性及其定量结合的能力应有助于鉴定和研究参与霍乱弧菌在人小肠上皮细胞定殖的黏附因子。
