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Actin polymerization in the mammalian cytosol can be locally activated by mechanisms that relieve the autoinhibited state of N-WASP, an initiator of actin assembly, a process that also requires the protein Toca-1. Several pathogenic bacteria, including Shigella, exploit this host feature to infect and disseminate efficiently. The Shigella outer membrane protein IcsA recruits N-WASP, which upon activation at the bacterial surface mediates localized actin polymerization. The molecular role of Toca-1 in N-WASP activation during physiological or pathological actin assembly processes in intact mammalian cells remains unclear. We show that actin tail initiation by S. flexneri requires Toca-1 for the conversion of N-WASP from a closed inactive conformation to an open active one. While N-WASP recruitment is dependent on IcsA, Toca-1 recruitment is instead mediated by S. flexneri type III secretion effectors. Thus, S. flexneri independently hijacks two nodes of the N-WASP actin assembly pathway to initiate localized actin tail assembly.

Shigella species cause diarrhoea by invading and spreading through the epithelial layer of the human colon. The infection triggers innate immune responses in the host that the bacterium combats by translocating into the host cell cytosol via a type 3 secretion system bacterial effector proteins that interfere with host processes. We previously demonstrated that interaction of the Shigella type 3 secreted effector protein IcsB with the host protein Toca-1 inhibits the innate immune response microtubule-associated protein light-chain 3 (LC3)-associated phagocytosis, and that IcsB interaction with Toca-1 is required for inhibition of this host response. Here, we show that Toca-1 in vitro precipitated not only IcsB, but also the type 3 secreted proteins OspC3, IpgD and IpaB. OspC3 and IpgD precipitation with Toca-1 was dependent on IcsB. Early during infection, most of these proteins localized near intracellular Shigella. We examined whether interactions among these proteins restrict innate host cell responses other than LC3-associated phagocytosis. In infected cells, OspC3 blocks production and secretion of the mature pro-inflammatory cytokine IL-18; however, we found that interaction of OspC3 with IcsB, either directly or indirectly via Toca-1, was not required for OspC3-mediated restriction of IL-18 production. These results indicate that interactions of the host protein Toca-1 with a subset of type 3 effector proteins contribute to the established function of some, but not all involved, effector proteins.

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