A273 THE CROHN'S DISEASE-ASSOCIATED PATHOBIONT ADHERENT-INVASIVE E. COLI (AIEC) INDUCES MITOCHONDRIAL FISSION IN EPITHELIAL CELLS IN ADVANCE OF APOPTOSIS. (1st March 2018) - Mancini, N
Abstract: Background: Adherent-invasive E. coli are a putative etiological agent in a cohort of patients with Crohn's disease. Mitochondrial dysfunction has been described in inflammatory bowel disease. Mitochondria are not distinct organelles; they exist as a dynamic network that constantly remodels via the processes of fission and fusion to meet the cells energy demands and allow recycling of damaged mitochondria. This is emerging as an area of interest in host-bacterial interaction but it is unknown if (and then how) infection with AIEC affects mitochondrial dynamics in epithelia and the consequences of this for the cell. Hypothesis : AIEC induce mitochondrial fragmentation in intestinal epithelial cells in an invasion-dependent manner that requires ROS, resulting in apoptosis and decreased epithelial barrier function. Aims: To explore the relationship between mitochondrial dynamics and epithelial function in vitro and determine if, then how, infection with AIEC affects mitochondrial dynamics and any consequence for epithelial barrier function. Methods: Human colon-derived epithelial lines were cultured with a non-invasive E. coli, AIEC (10 4 –10 8 cfu; 4-16h), fixed (dead) AIEC, or spent medium from bacterial cultures. Epithelia were examined: (a) ATP levels; (b) live-cell imaging of mitochondria morphology and membrane potential with confocal microscopy; (c) immunoblotting of whole cell protein extracts for the mitochondrial fusion protein Optic Atrophy Factor 1 (OPA1) and Dynamin-Related Protein 1 (Drp1); (d) ROS neutralized by the antioxidants; and, (f) measurement of barrier function and apoptosis. Results: AIEC infection resulted in reduced mitochondrial membrane potential and ATP, and dramatic mitochondrial fragmentation accompanied by OPA1 cleavage and recruitment of Drp1 to the mitochondria in gut epithelia in a time- and dose-dependent manner. The mitochondrial fragmentation was not reproduced by exposure to AIEC conditioned medium or fixed bacteria cells, and was not abrogated by treatment with a general (vitamin C) or a mitochondrial-specific (mitoTEMPO) anti-oxidant co-treatment. AIEC induced epithelial barrier loses, however preliminary trials with inhibitors of fission, Mdivi1 and P110, did not prevent the AIEC-induced drop in transepithelial resistance. Mitochondrial fragmentation preceded apoptosis in AIEC-infected epithelial cells. Conclusions: As a putative cause or contributor to Crohn's disease, AIEC drive massive mitochondrial fragmentation in model gut epithelia independent of ROS generation or AIEC soluble factors. Epithelial cell apoptosis is a feature of AIEC infection, which may be a consequence of excessive mitochondrial fission, that would be predicted to compromise epithelial barrier function, potentiating or reactivating inflammatory disease. Funding Agencies: CCC, CIHRNSERC, University of Calgary Eyes High Funding