Type 3 secretion systems (T3SSs) are conserved bacterial nanomachines that inject virulence proteins (effectors) into eukaryotic cells during infection. Due to their ability to inject heterologous proteins into human cells, these systems are being developed as therapeutic delivery devices. The T3SS assembles a translocon pore in the plasma membrane and then docks onto the pore. Docking activates effector secretion through the pore and into the host cytosol. Here, using Shigella flexneri, a model pathogen for the study of type 3 secretion, we determined the molecular mechanisms by which host intermediate filaments trigger docking and enable effector secretion. We show that the interaction of intermediate filaments with the translocon pore protein IpaC changed the pore’s conformation in a manner that was required for docking. Intermediate filaments repositioned residues of the Shigella pore protein IpaC that are located on the surface of the pore and in the pore channel. Restricting these conformational changes blocked docking in an intermediate filament-dependent manner. These data demonstrate that a host-induced conformational change to the pore enables T3SS docking and effector secretion, providing new mechanistic insight into the regulation of type 3 secretion.

Highlights d Shigella flexneri reduces membrane tension to promote intercellular spread d The type 3 secretion protein IpaC is required for efficient protrusion formation d IpaC interacts with b-catenin, altering the dynamics of adherens junctions

Type 3 secretion systems (T3SSs) are conserved bacterial nanomachines that inject virulence proteins (effectors) into eukaryotic cells during infection. Due to their ability to introduce heterologous protein into human cells, these systems are being developed as therapeutic delivery devices. The T3SS assembles a translocon pore in the plasma membrane and then docks onto the pore. Docking activates effector secretion through the pore and into the host cytosol. Here, using Shigella flexneri, a model pathogen for the study of type 3 secretion, we determined the molecular mechanisms by which host intermediate filaments trigger docking and enable effector secretion. We show that the interaction of intermediate filaments with the translocon pore protein IpaC changed the pore’s conformation in a manner that was required for docking. Intermediate filaments repositioned residues of the Shigella pore protein IpaC that are located on the surface of the pore and in the pore channel. Restricting these conformational changes blocked docking in an intermediate filament-dependent manner. These data demonstrate that a host-induced conformational change to the pore enables T3SS docking and effector secretion, providing new mechanistic insight into the regulation of type 3 secretion.Author summaryThe movement of bacterial proteins across membranes is essential for bacterial physiology and bacterial virulence. The type 3 secretion system moves bacterial virulence proteins from the inside of bacterial pathogens into human cells. To do so, the type 3 secretion system forms a pore in the plasma membrane of the target cell, attaches (docks) onto the pore, and delivers virulence proteins through the pore. Docking is essential for establishing a continuous channel from the inside of the bacteria to the inside of the human cell. What enables the type 3 secretion system to dock onto pores is not understood. We show that structural proteins in human cells, intermediate filament proteins, induce structural rearrangements to the type 3 secretion pore that trigger docking and that enable the subsequent delivery of virulence proteins into human cells. Due to the wide prevalence of type 3 secretion systems among human pathogens, these findings are likely to broadly enhance our understanding of type 3 secretion.

SummaryDuring infection, a subset of bacterial pathogens invades into the eukaryotic cytosol and spreads between cells of an epithelial layer. This intercellular spread is essential for disease and requires actin-based motility leading to the formation of plasma membrane protrusions. Protrusions are engulfed by the adjacent cell in an active process requiring both bacterial and eukaryotic proteins. Here, we demonstrate that the Shigella spp. type 3 secretion system protein IpaC promotes bacterial spread by reducing intercellular tension. S. flexneri producing a point mutant of IpaC that cannot interact with the cell-cell adhesion protein β-catenin were unable to reduce intercellular tension, form protrusions, or spread, demonstrating that interaction of IpaC with β-catenin is required for these processes. Spread was restored by chemical reduction of intercellular tension or genetic depletion of β-catenin. This work defines a molecular mechanism by which Shigella overcomes host cell-cell tension to mediate spread.