1:00 pm, Monday, 22 May
Room BIG13, Ground Floor
710 Cumberland St
Shigella flexneri is a gram-negative, enteric pathogen that causes dysentery in humans. Critical for pathogenesis is the ability of the bacterium to spread within the human colonic epithelium without rupturing the plasma membrane of host cells. Cell-to-cell spread of Shigella involves actin-based motility (ABM), which delivers bacteria to the plasma membrane of the human cell. Bacteria then deform this membrane into a finger-like projection called a "protrusion" that is internalized by a neighboring host cell, resulting in spread. Although the mechanism of ABM of Shigella is well understood, less is known about how this bacterium produces plasma membrane protrusions that mediate spread.
My study shows that Shigella subverts a host process called “polarized exocytosis” in order to enhance the generation of protrusions and cell-to-cell spread. Polarized exocytosis is the targeted fusion of intracellular vesicles to expand specific sites of the plasma membrane. This process is mediated by a human octameric protein complex known as the exocyst, GTPase regulators of the exocyst, and a membrane fusion machinery composed of SNARE proteins. Using an exocytic probe comprising the SNARE protein VAMP3 fused to EGFP, I found that polarized exocytosis is upregulated in Shigella protrusions in human cells. Furthermore, depletion of exocyst proteins, VAMP3, or the GTPase regulator RalA by RNA interference (RNAi) each significantly reduced protrusion formation efficiency and cell-to-cell spread of Shigella. Interestingly, depletion of exocyst proteins, VAMP3, or RalA also reduced the mean lengths of residual protrusions made by Shigella. Collectively these results indicate that the exocyst complex, VAMP3, and RalA are needed for both initiation and the elongation of Shigella protrusions. Finally, we demonstrate that stimulation of host exocytosis by Shigella requires the bacterium’s type three secretion system (T3SS), which is known to stimulate infection by injecting ~ 25 effector proteins into host cells. Collectively these results indicate that Shigella uses its T3SS to manipulate the function of the exocyst, resulting in enhanced protrusion formation and spread