Shigella protein complex-mediated repression of innate immunity early during infection

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Mammalian cells utilize multiple mechanisms to recognize and respond to invading bacteria. This includes inflammasome-mediated secretion of pro-inflammatory cytokines to promote recruitment of immune cells to the site of infection and LC3-associated phagocytosis of entering bacteria to promote pathogen elimination. Inflammasome components and LC3 are recruited to the human pathogen Shigella flexneri soon after entry into host cells, yet a subpopulation of intracellular bacteria avoids association with these markers. How this subpopulation escapes recognition by these innate immune factors is incompletely understood. We observed the subpopulation that does not associate with LC3 universally associates with the host protein Toca-1. We discovered that Toca-1 forms a complex with a small subset of proteins translocated into cells by S. flexneri via a type three secretion system. Among the ~30 proteins translocated by S. flexneri, I observed that Toca-1 specifically interacts with IpaB, IcsB, OspC3, and IpgD. We have shown that IcsB represses LC3-associated phagocytosis, and others have demonstrated that OspC3 inhibits caspase activation, raising the possibility that this complex functions in aggregate to inhibit innate immune responses. Specifically, I hypothesize that (1) the Toca-1, IcsB, IpgD, OspC3, and IpaB complex forms in a defined arrangement; (2) that the Toca-1-S. flexneri secreted protein complex is assembled on the vacuolar membrane or on vacuolar membrane fragments via the membrane-embedded translocon protein IpaB; and (3) that complex formation per se coordinates restriction of LC3-associated phagocytosis with OspC3-mediated inhibition of caspase activation. The following specific aims are designed to test aspects of this hypothesis: 1. To define the dependency of complex formation between Toca-1 and S. flexneri type three secreted proteins that precipitate with Toca-1. 2. To test whether the Toca-1-S. flexneri secreted protein complex per se restricts innate immune responses in host cells. This proposal will generate mechanistic insights into how S. flexneri evades the innate immune response in host cells early during infection. The results obtained in these studies are likely to have important implications for our understanding of the innate immune response to intracellular pathogens in general.