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

Abstract

To propel itself in infected cells, the pathogen Shigella flexneri subverts the Cdc42-controlled machinery responsible for actin assembly during filopodia formation. Using a combination of bacterial motility assays in platelet extracts with Escherichia coli expressing the Shigella IcsA protein and in vitro analysis of reconstituted systems from purified proteins, we show here that the bacterial protein IcsA binds N-WASP and activates it in a Cdc42-like fashion. Dramatic stimulation of actin assembly is linked to the formation of a ternary IcsA–N-WASP–Arp2/3 complex, which nucleates actin polymerization. The Arp2/3 complex is essential in initiation of actin assembly and Shigella movement, as previously observed for Listeria monocytogenes. Activation of N-WASP by IcsA unmasks two domains acting together in insertional actin polymerization. The isolated COOH-terminal domain of N-WASP containing a verprolin-homology region, a cofilin-homology sequence, and an acidic terminal segment (VCA) interacts with G-actin in a unique profilin-like functional fashion. Hence, when N-WASP is activated, its COOH-terminal domain feeds barbed end growth of filaments and lowers the critical concentration 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 to the bacterium surface. VASP is not involved in Shigella movement, and the function of profilin does not require its binding to proline-rich regions.

Neural Wiskott-Aldrich syndrome protein (N-WASP) is the specific ligand for Shigella VirG among the WASP family and determines the host cell type allowing actin-based spreading

“…It has been shown that human platelet extracts supported Shigella actin-based motility as added N-WASP in vitro (Egile et al, 1999). Recent reports using N-WASP-deficient mouse embryonic fibroblasts (Lommel et al, 2001;Snapper et al, 2001) confirmed our previous work indicating that N-WASP is required for Shigella motility in mammalian cells (Suzuki et al, 1998).…”

“…As shown in Fig. 3D, when wild-type N-WASP (0.1 mM) was added to the mixture in the presence of 20 nM Arp2/3 complex and 2.2 mM pyrene-actin, the actin polymerization activity was slightly stimulated, thus confirming that N-WASP alone is insufficient for stimulating actin polymerization by Arp2/3 complex (Egile et al, 1999;Rohatgi et al, 1999;Suzuki et al, 2000), whereas in the presence of NW1 (0.1 mM), the activity of NW1-Arp2/3 complex was markedly stimulated (about 2.5-fold; Fig. 3D).…”

“…It has been reported that N-WASP is required for generating the actin tail from motile S. flexneri (Suzuki et al, 1998;Egile et al, 1999). Arp2/3 complex is also required for actin-based motility of Shigella, in which N-WASP stimulates the actin nucleation activity of the Arp2/3 complex in vitro (Egile et al, 1999).In mammalian cells, five WASP family members, N-WASP, WASP and WAVE1-3, have been identified and characterized to date (Suetsugu et al, 1999); however, none of them except N-WASP has been elucidated for involvement in the actin-based motility of Shigella in infected cells. It has been shown that human platelet extracts supported Shigella actin-based motility as added N-WASP in vitro (Egile et al, 1999).…”

“…Arp2/3 complex is also required for actin-based motility of Shigella, in which N-WASP stimulates the actin nucleation activity of the Arp2/3 complex in vitro (Egile et al, 1999).…”

Spatial and temporal regulation of actin polymerization for cytoskeleton formation through Arp2/3 complex and WASP/WAVE proteins

“…The choice of activators of the Arp2/3 complex may also explain the different observations. N-WASP VCA and ActA have been reported to have profilin-like activity, where the complexes of VCA or ActA and actin monomers associate with barbed ends [Boujemaa-Paterski et al, 2001;Egile et al, 1999]. The different choice of capping pro- The concentrations of VCA, Arp2/3 complex, and actin monomer in the reaction mixture were 100 nM, 60 nM, and 2 M, respectively.…”