title: Elevation of intracellular free calcium levels in HEp-2 cells infected with enteropathogenic Escherichia coli.
abstract: Enteropathogenic Escherichia coli (EPEC) are a class of diarrheagenic organisms that induce a characteristic attaching and effacing lesion in enterocytes and various cultured cell lines. Infection of cultured HEp-2 cells by EPEC isolates 2036-80 (serotype O119) and E2348-69 (serotype O127) resulted in significant elevation of intracellular free calcium levels, determined quantitatively with the fluorescent calcium indicator dye 2-([2-bis(carboxymethyl)amino-5-methylphenoxy]methyl)-6-methoxy-8- bis(carboxymethyl)aminoquinoline. This effect, which was not observed on infection with non-lesion-forming E. coli strains, was inhibited by dantrolene, a drug that prevents calcium mobilization from intracellular stores. Moreover, activated protein kinase C in infected cells was dissociated from cell membranes by a process that was inhibited by cyclosporin A, suggesting involvement of the calcium-dependent protease calpain. A qualitative method for observing intracellular calcium fluxes by fluorescence microscopy with the recently described fluorescein-based indicator fluo-3 was used to screen a collection of well-characterized E. coli isolates from patients with infantile enteritis. Increased localized calcium-dependent fluo-3 fluorescence was observed only in HEp-2 cells infected with known lesion-forming EPEC strains. We propose that enhancement of intracellular free calcium levels in enterocytes infected with EPEC would result in formation of the characteristic lesion by calcium-dependent activation of actin-depolymerizing proteins, with eventual loss of absorptive capacity.
authors.affiliation: Department of Genetics, University of Leicester, United Kingdom.
authors.forename: T J
authors.initials: TJ
authors.lastname: Baldwin
authors.forename: W
authors.initials: W
authors.lastname: Ward
authors.forename: A
authors.initials: A
authors.lastname: Aitken
authors.forename: S
authors.initials: S
authors.lastname: Knutton
authors.forename: P H
authors.initials: PH
authors.lastname: Williams
mesh_terms: D002118:Calcium / Q000032:analysis*; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D004927:Escherichia coli Infections / Q000378:metabolism*; D007295:Inositol Phosphates / Q000378:metabolism; D011493:Protein Kinase C / Q000032:analysis
chemical_list: D007295:Inositol Phosphates; D011493:Protein Kinase C; D002118:Calcium