M Dytoc1, L Fedorko, P M Sherman. 1. Division of Gastroenterology, Hospital for Sick Children, Toronto, Ontario, Canada.
Abstract
BACKGROUND/AIMS: Human enteropathogenic Escherichia coli infection of epithelial cells is characterized by attaching and effacing adhesion. To determine if signal transduction responses are involved in this adhesion phenotype, levels of inositol 1,4,5-triphosphate and cytosolic free calcium were measured in tissue culture cells infected with enteropathogenic E. coli strain E2348 (serotype O127:H6). METHODS: Inositol triphosphate levels were measured by using a commercial binding assay, and intracellular calcium levels were determined by spectrofluorometry. RESULTS: Elevated levels of both inositol triphosphate (182% +/- 52%; P < 0.05) and intracellular calcium (125% +/- 40%, mean +/- SE; P < 0.05) were seen after infection of HEp-2 cells with strain E2348. In contrast, inositol triphosphate and intracellular calcium levels were not elevated in HEp-2 cells infected with six E. coli strains that did not cause attaching and effacing lesions. Subcellular calcium localization using oxalate precipitation and electron microscopy showed calcium accumulation within the terminal web subjacent to regions of attaching and effacing adhesion. Depleting external calcium did not eliminate formation of attaching and effacing lesions, whereas treatment of HEp-2 cells with an intracellular calcium chelator prevented attaching and effacing lesions. CONCLUSIONS: Enteropathogenic E. coli infection elevates both inositol triphosphate and intracellular calcium levels in cultured epithelial cells.
BACKGROUND/AIMS: Humanenteropathogenic Escherichia coli infection of epithelial cells is characterized by attaching and effacing adhesion. To determine if signal transduction responses are involved in this adhesion phenotype, levels of inositol 1,4,5-triphosphate and cytosolic free calcium were measured in tissue culture cells infected with enteropathogenic E. coli strain E2348 (serotype O127:H6). METHODS:Inositol triphosphate levels were measured by using a commercial binding assay, and intracellular calcium levels were determined by spectrofluorometry. RESULTS: Elevated levels of both inositol triphosphate (182% +/- 52%; P < 0.05) and intracellular calcium (125% +/- 40%, mean +/- SE; P < 0.05) were seen after infection of HEp-2 cells with strain E2348. In contrast, inositol triphosphate and intracellular calcium levels were not elevated in HEp-2 cells infected with six E. coli strains that did not cause attaching and effacing lesions. Subcellular calcium localization using oxalate precipitation and electron microscopy showed calcium accumulation within the terminal web subjacent to regions of attaching and effacing adhesion. Depleting external calcium did not eliminate formation of attaching and effacing lesions, whereas treatment of HEp-2 cells with an intracellular calcium chelator prevented attaching and effacing lesions. CONCLUSIONS: Enteropathogenic E. coli infection elevates both inositol triphosphate and intracellular calcium levels in cultured epithelial cells.