Shigella spp., the leading cause of death associated with diarrheal illness, are intracellular pathogen that require a type 3 secretion system (T3SS) to invade host cells. The T3SS is highly conserved among enteric bacterial pathogens and is essential for pathogenesis; the loss of T3SS leads to an attenuation of virulence. During S. flexneri infection, the T3SS secretes the bacterial proteins IpaB and IpaC, which form a pore in the plasma membrane (the translocon pore) that is predicted to be hetero-oligomeric. S. flexneri attaches (“docks”) onto the pore and secretes virulence proteins (effectors) through the pore into the host cell. Interactions between IpaB and IpaC in a natively delivered translocon pore have been postulated but never demonstrated. IpaB and IpaC monomers have been shown to oligomerize in vitro under specific conditions, suggesting similar interactions may be required for translocon pore function in the context of infection. My preliminary data establish approaches to investigate the translocon pore proteins when they are natively delivered during S. flexneri infection of mammalian cells. These approaches have demonstrated that the conformation of the translocon pore is dynamic and have identified specific IpaC residues that are differentially positioned in pores that are incompetent for docking and pores that are competent for docking. How IpaB is involved in the shift from a docking incompetent state to a docking competent state and the implications of IpaB involvement on pore organization are unknown. My overall hypothesis is that a functional translocon pore is formed when IpaB and IpaC oligomerize through interactions in their transmembrane spans in such a way that IpaB lines the majority of the pore channel, and that these interactions change during shifts in pore conformation. I propose to test aspects of this hypothesis with the following aims: (I) Determine the topology of IpaB in the context of natively-delivered translocon pores. (II) Define the organization of and interactions among IpaB monomers and between IpaB and IpaC in the context of natively delivered translocon pores. Using S. flexneri T3SS as a model, this proposal is highly likely to generate mechanistic insights into how the translocon pore contributes to T3SS function. Due to the highly conserved nature of the T3SS and its near ubiquitous requirement for the virulence of gram-negative enteric pathogens, the work proposed here is highly likely to have a broad impact on our understanding of bacterial pathogenesis. These studies will provide me with new expertise in molecular biology and protein biochemistry and place me in an ideal position for reaching my career goal of becoming an independent researcher.