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 0119) and E2348-69 (serotype 0127) resulted in significant elevation of intracellular free calcium levels, determined quantitatively with the fluorescent calcium indicator dye 2-{[2-bis (carboxymethyl)amino-5-methylphenoxyjmethyl}-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.

Involvement of calcium in interactions between gingival epithelial cells and Porphyromonas gingivalis

“…Thus, both the thapsigargin findings along with the BAP-TA and EDTA results indicate that the release of Ca2+ from the intracellular store is an important step in the invasion process. Similar conclusions were reached by Baldwin et al [7], who found that dantrolene, an inhibitor of Ca2+ release from the intracellular store, inhibited EPEC invasion of epithelial cells. Hence, it would appear that Ca2+ release from the intracellular store is important in the invasion process of both EPEC and P. gingivalis.…”

“…[Ca2+] changes in Henle-407 cells utilized a 3 h incubation on ice [6], and measurements of EPEC infection of HEp-2 cells occurred over l-6 h [7,14]. Hence, the overall frequency of P. gingivalisinduced [Ca2+] flux may actually be higher than we observed in 1 h.…”

“…Intracellular calcium plays an important role in a variety of eukaryotic signaling pathways including those that regulate microfilament activity [5]. Invasive pathogens such as enteropathogenic Escherichia coli (EPEC) and Salmonella typhimurium are capable of causing an elevation of intracellular calcium ion concentration ([Ca2+]) within epithelial cells [6,7]. Such calcium ion fluxes are important in the signalling events directed by these bacteria.…”

EspA, a protein secreted by enteropathogenic Escherichia coli, is required to induce signals in epithelial cells

“…EPEC signal transduction involves activation of host-cell tyrosine kinase activity leading to tyrosine phosphorylation of a 9OkDa host membrane protein, Hp90, and fluxes of intracellular inositol phosphate (IP3) and calcium (Baldwin etal., 1991;Foubister etal., 1994b;Dytoc et a/., 1994;Rosenshine et a/., 1992). Following these events, EPEC adheres intimately to the surface of the epithelial cell, accompanied by damage to host-cell microvilli and accumulation of cytoskeletal proteins beneath the adherent organism (Knutton etal., 1989;1987;Baldwin et a/., 1991;Finlay et al, 1992). A 35kb locus in EPEC, designated the LEE (!ocus of enterocyte effacement), encodes all of the known factors required for attaching and effacing lesion formation (McDaniel et al, 1995).…”

Mechanisms of diarrhoea

“…This phosphorylation leads to alterations in cell shape and in actin organisation (Knutton et al, 1989). EPEC infection also leads to raised intracellular calcium concentrations, and it seems likely that the formation of the characteristic lesion is due to calcium activation of the actin depolymerizing protein villin, leading to accumulation of actinomyosin within the lesion (Baldwin et al, 1991). At present it is unclear whether raised intracellular calcium concentrations also lead to phosphorylation of other cell membrane proteins involved in electrolyte transport across the brush border membrane.…”