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]iare not required for A/E lesion formation by EPEC and EHEC.

The Annexin A2/p11 complex is required for efficient invasion ofSalmonella Typhimurium in epithelial cells

“…What role this plays in Salmonella invasion is unclear. Both Salmonella and EPEC cause rapid increases in intracellular Ca 2+ when they interact with epithelial cells but it has not been conclusively demonstrated that this is required for invasion or pedestal formation (Ruschkowski et al, 1992;Pace et al, 1993;Bain et al, 1998;Gewirtz et al, 2000;Brown et al, 2008;Figueiredo et al, 2009). Recent experiments with Shigella, which invades via a similar process to Salmonella, have shown that a localized, rather than global, Ca 2+ response may be important in invasion (Tran Van Nhieu et al, 2013).…”

EPEC-activated ERK1/2 participate in inflammatory response but not tight junction barrier disruption

“…Gewirtz et al (10) have recently demonstrated that S. typhimurium-induced IB␣ degradation in intestinal epithelial cells is Ca 2ϩ dependent. Although the published results have been conflicting, EPEC has been shown to increase intracellular Ca 2ϩ in eukaryotic cells (2)(3)(4). We therefore measured the concentration of intracellular Ca 2ϩ in uninfected control T84 cells and cells infected with EPEC for 30 min.…”

Enteropathogenic and enterohaemorrhagic Escherichia coli : more subversive elements

“…Now, new data have raised serious doubts about any role for calcium signalling in A/E lesion formation. Bain et al (1998) used calcium imaging fluorescence microscopy, which allows both temporal and spatial measurements of intracellular calcium concentration ([Ca] i ) in EPEC/EHEC-infected epithelial cells to be determined. Not only were these workers unable to demonstrate localized increases in [Ca] i at sites of A/E EPEC and EHEC adhesion, they were unable to detect any significant alterations in cell calcium in infected compared with uninfected cells.…”