The intestinal inflammatory response is mediated in large part by synthetic upregulation of secreted cytokines and other inflammatory effector molecules. These inflammatory mediators are activated at the transcriptional level by the action of DNA binding transcription factors such as NF-kB. NF-kB itself is activated by a rapid post translational pathway involving sequential phosphorylation, ubiquitination and degradation of the inhibitor molecule, IkB. Unique among tissues, the intestinal epithelia functions in close contact with a diverse prokaryotic flora. There is increasing interest in the effects, both deleterious and beneficial, of these bacteria on the host epithelia. In this proposal, we describe enteric commensal bacterial strains that repress activation of the key NF-kB regulator by influencing a rate-limiting biochemical step, ubiquitination. Ubiquitination is an inducible covalent modification with a well defined role in protein turnover and degradation, and emerging roles in signal transduction, endocytic trafficking and other cellular functions. Bacteria and their products may influence covalent modifications necessary for activation of the enzymes responsible for ubiquitination of IkB and potentially other proteins involved in epithelial signaling. In other work, we have identified candidate effector molecules in enteric pathogens that are potent inhibitors of NFkB and likely act as deubiquitinating enzymes. We hypothesize these proteins will have profound effects on eukaryotic proinflammatory pathways and control of cellular apoptosis. ? ? Our overall hypothesis is that enteric bacteria, both commensal inhabitants of the intestine and known pathogens, have evolved mechanisms to modulate epithelial signaling pathways by influencing the eukaryotic ubiquitin system. Depending the specific biological context, the consequences of these interactions may by causal of infectious and idiopathic colitis, and/or may contribute to the physiology and overall health of the intestine. This proposal describes a variety of in vitro and in vivo experimental methods to study how bacteria influence the ubiquitination system in the intestinal epithelia, especially as it relates to the activation of the NF-kB and apoptotic pathways. Elucidation of these host-microbe interactions will increase our understanding of the epithelial and bacterial factors involved in human enterocolitis, and potentially in idiopathic inflammatory intestinal disease. ? ? ?
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