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Shigella, the causative agent of bacillary dysentery, is capable of directing its movement within host cells by forming an actin comet tail. The VirG (IcsA) pro-tein expressed at one pole of the bacterium recruits neural Wiskott-Aldrich syndrome protein (N-WASP), a member of the WASP family, which in turn stimulates actin-related protein (Arp) 2/3 complex-mediated actin polymerization. As all the WASP family proteins induce actin polymerization by recruiting Arp2/3 complex, we investigated their involvement in Shigella motility. Here, we show that VirG binds to N-WASP but not to the other WASP family proteins. Using a series of chimeras obtained by swapping N-WASP and WASP domains, we demonstrated that the specificity of VirG to interact with N-WASP lies in the N-terminal region containing the pleckstrin homology (PH) domain and calmodulin-binding IQ motif of N-WASP. A conformational change in N-WASP was important for the VirG-N-WASP interaction, as elimination of the C-terminal acidic region, which is responsible for the intramolecular interaction with the central basic region of N-WASP, affected the specific binding to VirG. We observed that, in haematopoietic cells such as macrophages, polymorphonuclear leucocytes (PMNs) and platelets, WASP was predominantly expressed, whereas the expression of N-WASP was greatly suppressed. Indeed, unlike Listeria, Shigella was unable to move in macrophages at all, although the movement was restored as N-WASP was expressed ectopically. Thus, our findings demonstrate that N-WASP is a specific ligand of VirG, which determines the host cell type allowing actin-based spreading of Shigella.