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019 -CAG STUDENT PRIZE A Flynn, N Vergnolle, A Buret AIM: The attaching and effacing (A/E) organism enteropathogenic E. coli (EPEC) is an important cause of diarrhea in developing countries. Our aim was to evaluate the mechanisms whereby EPEC and Citrobacter rodentium (murine EPEC) might disrupt tight junctions and induce epithelial apoptosis.
Inflammation Research Network, University of Calgary, Calgary, Alberta
METHODS: In vivo: Mice were infected via intragastric gavage with 5*108 C. rodentium colony forming units. Intestinal permeability was assessed via the lumenal-to-blood passage of 51Cr-labeled EDTA. Colonization and inflammation were assessed by plating and MPO activity assays. In vitro: Small intestinal SCBN cells were treated with purified E. coli LPS. Mouse colorectal carcinoma CMT-93 cells and human T84 cells were exposed to C. rodentium and EPEC, respectively. Permeability was measured via the paracellular translocation of a FITC-conjugated dextran probe. Immunofluorescence was performed to assess tight junctional disruption. For assessment of apoptosis, cells were processed via a Cell Death ELISA or immunoblotted for cleaved PARP.
RESULTS: C. rodentium caused increased intestinal permeability in mice. Maximal permeability occurred prior to maximal colonization and inflammation, and was associated with the disruption of claudins-4 and -5. Mice deficient in TLR-4-/- succumbed to the infection, demonstrating increased colonization and mortality, and decreased MPO activity. SCBN cells treated with high levels of LPS became apoptotic and displayed caspase-3-dependent increases in paracellular permeability. CMT-93 and T84 cells demonstrated increased Rho kinase activity and apoptosis upon exposure to C. rodentium or EPEC, respectively. Infected cells also displayed increased paracellular permeability that was independent of caspase-3 activity, but was dependent on Rho kinase. Interestingly, C. rodentium-induced CMT-93 cell apoptosis was caspase-independent, while EPEC-induced T84 cell apoptosis was caspase-dependent. Immunofluorescence confirmed the caspase-3-independent disruption of claudins-4 and -5 by the (A/E) pathogens.
CONCLUSION: A/E pathogens may induce host epithelial malfunction characterized by apoptosis and increased permeability. The host and pathogen signals involved in these responses are diverse, and may be species-specific.