The deadly enteropathogen Shigella flexneri usurps components of the actin cytoskeleton for entry and movement in the host cell, and for the formation of protrusions used to infect neighboring cells. The Shigella protein IpaA is secreted from the bacterium upon cell contact and is essential for bacterial uptake and pathogenesis. IpaA accomplishes these tasks by binding to vinculin, a highly conserved cytoskeletal protein that plays essential roles in directing the assembly of actin networks following the formation of focal (cell- matrix) and adherens (cell-cell) junctions. Vinculin normally orchestrates changes in the actin cytoskeleton by binding to other key partners such as talin, which binds to integrin receptors in focal adhesions, or to a- actinin, which plays key roles in adherens junctions. Our recent studies have shown that talin and a-actinin activate vinculin by disrupting the intramolecular hydrophobic interactions of vinculin's tail domain (Vt) with its AMerminal seven-helical bundle (Vh1) domain, by provoking remarkable and unique changes in the structure of this domain through a process we have coined helical bundle conversion. IpaA binding to vinculin has been shown to increase its binding affinity for F-actin and to depolymerize actin filaments, and our new studies have shown that this occurs through IpaA's ability to specifically augment a new function for vinculin in capping the barbed ends of actin filaments. Importantly, our Preliminary Studies have also shown that IpaA disrupts the Vh1-Vt interaction, establishing the mechanism by which Shigella activates vinculin, and that IpaA also disrupts the association of vinculin with talin and a-actinin. These studies support a model whereby IpaA binding provokes unique changes in the structure of vinculin that disrupt the formation of adherens junctions and focal adhesions, subverting vinculin to carry out functions essential for Shigella pathogenesis. To precisely define the mechanism of activation of vinculin by IpaA, experiments in Specific Aim #1 will define the crystal structure of the vinculin:lpaA complex. Our new studies have shown that the binding of talin and a-actinin to vinculin's Vh1 domain is sufficient to trigger unique conformational changes throughout the whole molecule. We therefore hypothesize that IpaA binding also induces unique structural changes in full-length vinculin, and to test this notion the experiments in Specific Aim #2 will determine the effects of IpaA on the conformation of full-length vinculin and how it alters its associations with its binding partners. Finally, disrupting the interactions of vinculin with IpaA should have profound effects on Shigella pathogenesis. To address this hypothesis, experiments in Specific Aim #3 will determine the effects of disrupting the vinculin-lpaA interaction on Shigella entry, motility and spread, and upon Shigella pathogenesis in vivo. The proposed experiments should define the interactions of IpaA and vinculin that are required to direct Shigella pathogenesis, and it is hoped that these studies will also provide the foundation for new strategies to combat this deadly pathogen.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
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Macromolecular Structure and Function B Study Section (MSFB)
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Mills, Melody
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Scripps Research Institute
La Jolla
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