Abstract
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. A key unresolved question is whether the formation of these protrusions involves bacterial exploitation of host processes apart from actin polymerization.
Here, I demonstrate 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 and that this upregulation depends on the host exocyst complex. The exocyst complex contains eight proteins: Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70, and Exo84. Importantly, depletion of Sec3, Sec6, Sec8, Sec10, or Exo70 by RNA interference (RNAi) each significantly reduced protrusion formation efficiency and cell-to-cell spread of Shigella. RNAi-induced depletion of the SNARE protein VAMP3 or the GTPase regulator RalA caused similar inhibitions in bacterial protrusion formation and spread. Interestingly, depletion of exocyst proteins, VAMP3, or RalA not only impaired the frequency of protrusion formation but also reduced the mean lengths of residual protrusions made by Shigella. Collectively these results indicate that the exocyst complex, VAMP3, and the exocyst regulator RalA are needed for both initiation and the elongation of Shigella protrusions.
Shigella infects human cells by using a Type III secretion system (T3SS) to inject ~ 25 bacterial effector proteins into the host cell cytosol. Using a Shigella strain that inducibly expresses the T3SS structural components MxiM, IpaB, IpaC, or IpaD, I found that stimulation of host exocytosis requires the Shigella T3SS. Moreover, the exocyst component Exo70 and regulator RalA were recruited to Shigella protrusions, and this recruitment depended on the T3SS. Finally, inhibition in expression of the T3SS components MxiM, IpaB, IpaC, or IpaD impaired protrusion formation and cell-to-cell spread of Shigella. Collectively, these results indicate that Shigella uses its T3SS to manipulate the function of the exocyst, resulting in enhanced protrusion formation and spread. Future work with Shigella should focus on identifying the specific T3SS effectors that target the exocyst or exocyst regulators to promote intercellular spread.