Molecular Mechanisms of Modulation of N-WASP activity

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DESCRIPTION (provided by applicant): Shigella sp. cause 550 million cases of diarrhea or dysentery and 1.1 million deaths annually worldwide. S. flexneri are intracytoplasmic gram negative bacteria which cause disease by invasion and spread through the colonic mucosa. Survival and spread of S. flexneri are mediated by bacterial manipulation of the host cytoskeleton. In the host cell cytoplasm, bacteria initiate nucleation and polymerization of actin filaments at one pole, forming a propulsive actin tail. The bacterial outer membrane protein IcsA is required for actin tail assembly in conjunction with host factors including actin nucleator Arp 2/3, actin nucleation promoting factor N-WASP, and Toca-1. In resting cells, N-WASP is maintained in an autoinhibited conformation and is activated by Cdc42 in conjunction with Toca-1. Activation of N-WASP during S. flexneri actin tail assembly is independent of Cdc42, and we have recently shown that N-WASP activation requires Toca-1. Our studies indicate that Toca-1 plays a role in relieving autoinhibition of N-WASP during actin tail formation. Once activated, N-WASP associated with S. flexneri does not require the presence of Toca-1 for ongoing actin polymerization. N-WASP associated with S. flexneri shows shifted migration on SDS-PAGE compared to N-WASP from cell lysate, suggesting that a covalent modification has been induced by S. flexneri. The goals of my research are to: I) Characterize the modification made to N-WASP by S. flexneri and determine the effect of this modification on N-WASP conformation and actin polymerization, II) Determine the effect of the modification to N-WASP on cell motility and invasion and III) Identify the bacterial factor responsible for making this modification to N-WASP. Understanding mechanisms by which S. flexneri modulates the host cytoskeleton will generate insights into the mechanisms of bacterial manipulation of the host cytoskeleton relevant to Shigella pathogenesis and perhaps to the pathogenesis of other organisms as well. Moreover, because N-WASP activity is highly regulated by the cell, the results of the proposed studies are likely to lead to insights into the fundamental mechanisms of cellular modulation and control of actin polymerization. PUBLIC HEALTH RELEVANCE: Many bacteria that cause disease infect human cells, sometimes modifying structural proteins that give cells shape. My research proposal aims to identify mechanisms used by the bacterium Shigella flexneri to modify these structural proteins. Understanding processes by which bacteria modify these proteins can elucidate new avenues for developing vaccines and treatment strategies