Shigella IcsA Activation of Actin Assembly by N-WASP

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DESCRIPTION (provided by applicant) Shigella spp. are bacterial pathogens that cause an estimated I million deaths and 163 million cases of diarrhea and dysentery annually worldwide, predominantly in children. Shigella cause disease by invading and spreading through the colonic epithelium. Shigella spread by entering the cytoplasm of the host cell and inducing the assembly of an actin tail on the bacterial body. Actin tail formation involves bacterial recruitment of cellular actin assembly machinery. Dr. Goldberg laboratory has demonstrated that the Shigella protein IcsA is sufficient to mediate this process. IcsA utilizes the cellular Cdc42 signal transduction pathway for actin assembly, which has been shown to be important in filopodia formation. This cellular pathway involves Cdc42 activation of members of the Wiskott-Aldrich syndrome protein (WASP) family. Activated WASP family members activate the Arp213 complex to nucleate actin and polymerize new filaments, which Arp2/3 cross-links at 700 angles to pre-existing filaments. IcsA is known to bind N-WASP, a member of the WASP family, yet the mechanism by which it activates N-WASP is unknown. IcsA is essential to Shigella virulence. We propose to characterize the mechanism of lcsA activation of N-WASP. We will identify residues of N-WASP and lcsA that are important to either lcsA binding or lcsA activation of N-WASP. We will then test whether N-WASP is activated by lcsA in a fashion similar to its activation by Cdc42. These studies will provide insight into both the molecular mechanisms of Shigella pathogenesis and the molecular mechanisms of 0dc42-mediated actin assembly.