Enterohemorrhagic E. coli (EHEC) has emerged as an important agent of diarrheal disease and the leading cause of pediatric renal failure in the U.S. Intimate attachment to host cells is an essential step during intestinal colonization by EHEC. After initial host cell attachment, the bacterium injects into the host cell a number of molecules that trigger signaling pathways and result in the disruption of the eukaryotic cytoskeleton. Among the injected proteins is Tir, a protein that becomes localized in the host cell membrane and acts as a receptor for the bacterial outer membrane protein intimin. Intimin, encoded by the eae gene, is required for the formation of a highly organized for the formation of a highly organized cytoskeletal structure containing filamentous actin directly beneath the bound bacterium that lifts the bacterium above the plane of the host cell membrane on a """"""""pedestal"""""""". Deletion mutants of eae, which cannot induce the formation of this pedestal, are deficient for intestinal colonization. Thus, we postulate that Tir-intimin interaction is an essential early event in the development of disease caused by EHEC. We have identified regions of intimin and Tir that interact with each other , and have shown that the Tir-binding region of intimin is sufficient to induce actin condensation after pre-infection of host cells with E. coli. A detailed understanding of Tir-intimin binding, as well as of the molecular signals immediately downstream of this interaction, are required to gain insight into how EHEC colonizes the intestine and promotes damage. Thus, the following questions will be addressed: 1. What is the topological map of Tir in the eukaryotic membrane? 2. Is Tir binding by intimin sufficient to trigger actin condensation on preinfected cells? Latex beads that artificially bind TIR will be tested for the ability to induce actin condensation on preinfected eukaryotic cells. 3. How does intimin and Tir recognize each other? Genetic and biochemical approaches, including crystallographic studies, will be pursued to understand the molecular basis for this interaction. 4. Is Tir-intimin interaction essential to promote intestinal colonization? Point mutations in eae and tir that disrupt or restore Tir-intimin binding will be tested for their effect on colonization in an animal model for EHEC infection. 5. What mammalian cell factors interact with the cytoplasmic region(s) of Tir? Mammalian cell factors that directly receive from Tir the biochemical signal for actin filament formation will be identified. The proposed experiments may provide novel targets for therapeutic intervention during EHEC infection, as well as provide insight into the general cellular mechanisms by which actin assembly controlled.
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