Activation of the CDC42 effector N-WASP by the Shigella flexneri IcsA protein promotes actin nucleation by Arp2/3 complex and bacterial actin-based motility

To propel itself in infected cells, the pathogen Shigella flexneri subverts the Cdc42-controlled machinery responsible for actin assembly during filop odia formation, Using a combination of bacterial motility assays in platele t extracts with Escherichia coli expressing the Shigella IcsA protein and i n vitro analysis of reconstituted systems from purified proteins, we show h ere that the bacterial protein IcsA binds N-WASP and activates it in a Cdc4 2-like fashion. Dramatic stimulation of actin assembly is linked to the for mation of a ternary IcsA-N-WASP-Arp2/3 complex, which nucleates actin polym erization. The Arp2/3 complex is essential in initiation of actin assembly and Shigella movement, as previously observed for Listeria monocytogenes, A ctivation of N-WASP by IcsA unmasks two domains acting together in insertio nal actin polymerization, The isolated COOH-terminal domain of N-WASP conta ining a verprolin-homology region, a cofilin-homology sequence, and an acid ic terminal segment (VCA) interacts with G-actin in a unique profilin-like functional fashion. Hence, when N-WASP is activated, its COOH-terminal doma in feeds barbed end growth of filaments and lowers the critical concentrati on at the bacterial surface. On the other hand, the NH2-terminal domain of N-WASP interacts with F-actin, mediating the attachment of the actin tail t o the bacterium surface. VASP is not involved in Shigella movement, and the function of profilin does not require its binding to proline-rich regions.