Actin-based motility of intracellular Shigella flexneri

Actin polymerization in the mammalian cytosol is globally inhibited, but can be locally activated by N-WASP, an actin nucleation promoting factor. In resting cells, the majority of N-WASP is bound to another cellular protein, WIP, which stabilizes N-WASP in an autoinhibited conformation. Activation of N-WASP requires the relief of this autoinhibited conformation, a process dependent on at least two other proteins, the Rho GTPase Cdc42 and Toca-1. Shigella , an intracellular pathogen, has evolved to exploit this mammalian actin polymerization machinery to spread within and across infected cells. The Shigella outer-membrane protein IcsA recruits N-WASP to the bacterial surface, leading to localized actin polymerization. However, the mechanism of N-WASP activation at the bacterial surface was unclear. In this thesis, I investigate the mechanisms through which Shigella triggers actin polymerization, in particular the roles of Toca-1 and WIP in Shigella activation of N-WASP. I show that Toca-1 promotes Shigella actin assembly by relieving N-WASP autoinhibition on the bacterial surface. While N-WASP is recruited to the bacterial surface by IcsA, Toca-1 recruitment is instead mediated by Shigella type III secretion effectors. Thus, S. flexneri independently hijacks two distinct nodes of the N-WASP actin assembly pathway, N-WASP and Toca-1. Toca-1 is transiently required for the initiation of actin tail formation, but is not required for on-going actin polymerization once N-WASP is activated. In contrast, WIP inhibits Shigella actin assembly by inhibiting the relief of N-WASP autoinhibition. Based on my results, I propose a revised model of Shigella actin tail assembly: Shigella IcsA recruits autoinhibited N-WASP to one bacterial pole. WIP is displaced from N-WASP, by unclear mechanisms. Then, Toca-1, which is recruited to the bacterium by Shigella type III secreted effectors, activates N-WASP by relieving N-WASP autoinhibition, allowing N-WASP to activate actin polymerization. Once actin polymerization is initiated, Toca-1 dissociates from the bacterial surface, via a yet unknown mechanism, and is no longer required to maintain N-WASP in the open, active conformation. My findings further suggest that activation of N-WASP by cellular factors may generally involve a two step process, in which first WIP inhibition of N-WASP is removed, then Toca-1 relieves N-WASP autoinhibition.