Shigella are gram negative enteric pathogens that cause severe diarrheal disease and have been classified as a Category B Biological Agent. Shigella pathogenesis requires bacterial invasion of the colonic epithelium and bacterial spread through the colonic mucosa. Shigella entry into epithelial cells is mediated by effector molecules, secreted through a type III secretion apparatus, that activate Rho family GTPase signaling pathways to induce the formation of cell surface projections and membrane ruffles that engulf the bacteria by macropinocytosis. Both Cdc42 and Rac have been implicated as having a role in the Shigella entry process. Cdc42 is known to activate Rac; it is not clear whether Cdc42 involvement in Shigella entry is mediated exclusively via this link. Moreover, the downstream effectors of Cdc42 and/or Rac activation during Shigella entry are unknown. We have recently confirmed that the major Shigella pathway is Cdc42-dependent. However, we have also demonstrated the existence of a novel Cdc42-independent invasion pathway. Furthermore we have shown that the only known downstream effector of Cdc42 that activates the actin cytoskeleton, N-WASP, is not involved in Shigella entry. Once in the cytoplasm, Shigella moves by active assembly of an actin tail. Actin tail formation is mediated by the Shigella outer membrane protein IcsA, which binds and activates N-WASP. Activated N-WASP stimulates Arp2/3 complex-mediated actin assembly. The molecular mechanism by which IcsA binds and activates N-WASP is poorly understood. Our goals in this proposal are to: 1. Define the specific roles of Cdc42 and Rac in Shigella entry; 2. Identify and characterize the downstream effectors of Rho family activation during Shigella entry; and, 3. Elucidate the mechanism(s) by which Shigella IcsA activates N-WASP and determine whether this mechanism mimics Cdc42 activation of N-WASP These studies will define the specific cellular signaling pathways required for Shigella entry and actin tail formation and will identify downstream pathways of Rho family activation.
