INT-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.
A 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.
EAEC induced increase in [Ca2+]i seems to play a major role in host cytoskeletal F-actin rearrangements leading to invasion of the organism.
Our study undoubtedly will lead to an improved understanding of EAEC-pathogenesis.