Enteroaggregative Escherichia coli induced increase in intracellular calcium concentration modulates cytoskeletal F-actin rearrangement and bacterial entry in INT-407 cells

BackgroundEnteroaggregative Escherichia coli (EAEC) is an emerging enteric pathogen, associated with cases of acute and persistent diarrhoea worldwide. The pathogenesis of EAEC is yet to be understood. In intestinal epithelium, an increase in [Ca2+]i has been attributed due to the action of different enteric pathogens. EAEC was shown to increase [Ca2+]i in HEp-2 cells.The present study was undertaken to investigate the effect of EAEC induced increase in [Ca2+]i oncultured human intestinal epithelial cells.MethodsINT-407 cells were infected with EAEC (T8 strain) in the absence and presence of dantrolene (inhibitor of release of Ca2+ from intracellular stores)/verapamil (L-type Ca2+ channel blocker)/BAPTA-AM (Ca2+ chelator)/U73122 (PLC inhibitor)/Cytochalasin-D (inhibitor of actin polymerization). [Ca2+]i was estimated using Fura-2/AM. Cytoskeletal rearrangement was assessed by F-actin staining using TRITC-phalloidin. The invasiveness of EAEC-T8 to INT-407 cells was checked by electron microscopy and invasion assay.ResultsA significant increase in [Ca2+]i was observed in EAEC-T8 infected INT-407 cells, which was reduced in presence of dantrolene/verapamil/U73122. EAEC-T8 could induce cytoskeletal F-actin polymerization in INT-407 cells and was found to be invasive in nature. The cytoskeletal rearrangement as well as invasion of EAEC-T8 was attenuated in presence of U73122/dantrolene/BAPTA-AM/verapamil/cytochalasin D.ConclusionsEAEC induced increase in [Ca2+]i seems to play a major role in host cytoskeletal F-actin rearrangements leading to invasion of the organism.General significanceOur study undoubtedly will lead to an improved understanding of EAEC-pathogenesis.

► We studied the effect of EAEC induced increase in [Ca2+] i on INT-407 cells.
► A biphasic increase in [Ca2+]i was observed in EAEC-T8 stimulated INT-407 cells.
► EAEC-T8 strain induced dramatic alteration in F-actin distribution.
► [Ca2+]i and actin polymerization is involved in the invasion of this organism.
► [Ca2+]i plays a role in host F-actin polymerization leading to invasion of EAEC.