Increased levels of intracellular calcium are not required for the formation of attaching and effacing lesions by enteropathogenic and enterohemorrhagic Escherichia coli

Abstract

Elevated concentrations of intracellular calcium ([Ca]i) have been implicated as an important signalling event during attaching and effacing (A/E) lesion formation by enteropathogenic Escherichia coli (EPEC). The highly localized nature of the cytoskeletal and cell surface alterations occurring during A/E lesion formation suggests that there should be equally localized EPEC-induced signalling events. To analyze further the calcium responses to infection of HEp-2 cells by EPEC, we employed calcium-imaging fluorescence microscopy, which allows both temporal and spatial measurements of [Ca]i in live cells. Using this imaging technique, not only were we unable to detect any significant elevation in [Ca]i at sites of A/E EPEC adhesion, but, with several different classical EPEC and enterohemorrhagic E. coli (EHEC) strains and three different infection procedures, each of which resulted in extensive A/E bacterial adhesion, we were unable to detect any significant alterations in [Ca]i in infected cells compared to uninfected cells. In addition, chelation of intracellular free calcium with bis-(aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid (BAPTA) did not, as previously reported, prevent A/E lesion formation. We conclude that increased [Ca]i are not required for A/E lesion formation by EPEC and EHEC.

FIG. 1

FAS tests performed at the end of typical calcium-imaging experiments. The micrographs show fluorescence (left panels) and corresponding phase-contrast images (right panels) of HEp-2 cell monolayers spindown infected with EPEC strain E2348/69 for 1 h (A), gradually infected with EPEC strain E2348/69 for 6 h (B), gradually infected with EHEC strain E29962 for 6 h (C), and treated with BAPTA and then spindown infected with EPEC strain E2348/69 for 1 h (D). Each infection procedure resulted in good colonization of cells with A/E EPEC (A and B), although adhesion by EHEC strains was significantly less than that by EPEC strains (C); characteristic EPEC-induced actin accretion occurred in BAPTA-treated cells (D). Magnification, ×320.

FIG. 2

Montage of pseudocolor ratio images taken at 2-min intervals, showing [Ca]_i in HEp-2 cells spindown infected with E2348/69 for 1 h. The images show no significant alterations in [Ca]_i during the period of A/E lesion formation (images 5 to 10). After 40 min (image 21), 100 mM histamine was added to the cells, resulting in a large increase in [Ca]_i.

FIG. 3

Mean [Ca]_i (n = 4) in control HEp-2 cell monolayers and HEp-2 cell monolayers infected for 1 h with EPEC strain E2348/69 by the spindown and settledown procedures. No significant alteration in [Ca]_i was seen throughout the 1-h infection period, during which extensive A/E adhesion occurred. An ∼10-fold increase in [Ca]_i occurred in control cells following the addition of 1 μM ionomycin, confirming the responsiveness of the cells and the detection system.

FIG. 4

Mean [Ca]_i (n = 2) in control HEp-2 cell monolayers and HEp-2 cell monolayers infected for up to 6 h with four different EPEC strains and two different EHEC strains by using the gradual infection procedure. Values are presented as percentages of the mean [Ca]_i in the cells at time zero. No significant change in [Ca]_i was seen with any of the six strains over the 6-h time course. Error bars are illustrated for strain E2348/69 and are typical of those for the remainder of the strains.

FIG. 5

Effect of BAPTA on histamine-induced rises in HEp-2 cell [Ca]_i. Histamine (H) (1 mM) was added to both control and BAPTA-treated cells after 7 and 18 min (arrows). Histamine-stimulated rises in [Ca]_i were completely abrogated in the BAPTA-treated cells, whereas considerable increases in [Ca]_i were seen in the untreated cells.

FIG. 6

Mean percent cell death (n = 2) in HEp-2 cell monolayers gradually infected with EPEC strain E2348/69 for 6 h. Less than 5% cell death occurred over the 6-h time period of the experiment.