HOME
Return to Table of Contents
APOPTOSIS-INDUCING FACTOR CONTRIBUTES TO ENTEROPATHOGENIC E. COLI-INDUCED EPITHELIAL CELL APOPTOSIS
AN Flynn, AG Buret
Inflammation Research Network and Department of Biological Sciences, University of Calgary, Calgary, Alberta
The important diarrheagenic human pathogen enteropathogenic E. coli (EPEC) induces apoptosis in host epithelial cells. Previous research has demonstrated that bacterial EspF and the intrinsic and extrinsic apoptotic pathways are involved in this process. However, the roles of further apoptogenic factors await investigation, including apoptosis-inducing factor (AIF), a mitochondrial protein released into the cytosol and nucleus during certain forms of caspase-dependent and -independent apoptosis.
METHODS: Human intestinal epithelial T84 cells and human cervical epithelial HeLa cells were apically infected by EPEC strain E2348/69 or an espF mutant at a multiplicity of infection of 100:1. The release of AIF from mitochondria was measured by subcellular fractionation and immunoblotting. Pan-caspase inhibition was achieved using 50 µM Z-VAD. Apoptosis was assessed by immunoblotting for cleaved PARP as well as using a Cell Death ELISA. Knockdown of AIF expression was performed in HeLa cells by RNAi.
RESULTS: EPEC infection caused the time-dependent cytosolic accumulation of AIF in T84 cells. Pan-caspase inhibition partially prevented the release of the mature 57 kDa protein, but not the 62 kDa or 67 kDa forms. E2348/69 and espF were equally effective at inducing the nuclear accumulation of AIF. Knockdown of AIF expression in HeLa cells protected them from EPEC-mediated apoptosis.
DISCUSSION: EPEC infection of epithelial cells leads to the mitochondrial release of AIF, which contributes to cellular apoptosis. While caspases are activated upstream of AIF release, the noted EPEC virulence factor EspF is not required for this effect. This may explain the residual pro-apoptotic effects of EPEC strains deficient in EspF production. Further research in this area will be important for the elucidation of signaling pathways involved in EPEC-induced epithelial cell apoptosis.