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1Biosciences Division, General Atomics, PO Box 85608, San Diego, CA 92186-9784, USA
2Department of Microbiology and Immunology, Oregon Health Sciences University, 3181 SW Sam Jackson Park Road, Portland, OR 97201-3098, USA
3Author for correspondence: Kathleen Richardson. Tel: +1 503 725 3855. Fax: +1 503 725 3888. e-mail: b7kr@odin.cc.pdx.edu
ABSTRACT
Vibrio cholerae must colonize the human small intestine to cause diarrhoeal disease. V. cholerae strains N16961 (El 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 or 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.
Present address: Biology Department, Portland State University, PO Box 751, Portland, OR 97207-0751, USA.
Present address: Max-Planck-Institut für Biologie, Abt. Infektions-biologie, Spemannstrasse 34, D 72076 Tübingen, Germany.
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