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.