Executive Summary : | The present study was undertaken to assess Enteroaggregative Escherichia coli (EAEC) induced apoptosis and cell cycle modulation in cultured human intestinal epithelial cells of different origin. INT-407 (human small intestinal epithelial cell line) as well as HCT-15 (human colon carcinoma cell line) cells were infected separately with an invasive strain of EAEC (EAEC-T8, clinical isolate), non-invasive prototype strain of EAEC (EAEC-042) and a plasmid cured variant of EAEC-T8 (EAEC-pT8). The time of infection and the incubation period of the infected culture were selected in view of apoptosis by propidium iodide staining. Maximum extent of apoptosis was noted in case of EAEC-T8 as compared to EAEC-042 and EAEC-pT8 in both the cell lines (as assessed by the expression of phosphatidylserine on the host cell surface and internucleosomal cleavage of the host cell DNA) indicating the importance of invasion as well as the plasmid of this organism in this process. The characteristic features of programmed cell death were also noted in EAEC infected cells by fluorescence microscopy and transmission electron microscopy. Further, EAEC induced upregulation of Bax and Bak, downregulation / unalteration of Bcl-2 and Bcl-XL, reduction in the mitochondrial transmembrane potential, release of cytochrome cfrom mitochondria into cytosol and activation of pro-caspase-9 and pro-caspase-3 resulting in DNA fragmentation in the intestinal epithelial cells were also observed. Anincreased expression of Fas receptor, activation of procaspase-8 and up regulation ofBid in these cells clearly indicated the involvement of extrinsic pathway in EAEC induced apoptosis. Moreover, the plasmid-borne membrane proteins of EAEC were found to have maximum involvement tothis processand the adhesin of the organism was found tobe a possible contributor.The wild type invasive strain of EAEC could alsoarrest maximum number of cells at the S phase and G2-M phase of the cell cycle in both the intestinal epithelial cell linesandthe plasmid-borne membrane proteins of EAEC were found to have maximum contribution to this process.The outcome of our study has undoubtedly led to an improved understanding of the basic mechanism underlying the pathogenesis caused by EAEC. |
Co-PI: | Dr. Anuradha Chakraborti, Professor, Post Graduate Institute of Medical Education and Research (PGIMER), Chandigarh, Dr. Shalmoli Bhattacharya, Assistant Professor, Post Graduate Institute of Medical Education and Research (PGIMER), Chandigarh |