Abstract
Shigella flexneri is a gram-negative bacterium responsible for life-threatening gastroenteritis in children under five. S. flexneri induces its internalization into the host intestinal epithelium and undergoes actin-based motility to form plasma membrane protrusions that mediate spread to adjacent cells. While the mechanism of actin-based motility by S. flexneri has been extensively studied, how these bacteria form protrusions is less well understood. Recently, our group found that S. flexneri induces host polarized exocytosis via the human exocyst complex to enhance protrusion formation. Stimulation of host exocytosis requires S. flexneri’s type III secretion system (T3SS), a needle-like apparatus that injects ~ 25 effector proteins into the host cytosol. The overall aim of my project was to identify S. flexneri T3SS effectors that induce exocytosis, and to determine how these effectors manipulate the exocyst. Using an exocytic probe, S. flexneri mutant strains deleted for T3SS effector genes were screened to identify effectors that contribute to exocytosis in protrusions. One of the T3SS effectors found to promote exocytosis is IpgD, which has phosphatase activity on the host membrane lipid phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. Using established confocal microscopy-based assays, I found that IpgD is needed for efficient protrusion formation and cell-to-cell spread of S. flexneri. Further experiments revealed that IpgD may manipulate exocyst function via the host GTPase Arf6, a known regulator of the exocyst. Experiments involving small interfering RNA (siRNA)-mediated depletion of Arf6 or chemical inhibition of human activators of Arf6, indicate that this GTPase promotes exocytosis in protrusions and intercellular spread of S. flexneri in an IpgD-dependent manner. To assess IpgD’s role in activating Arf6 in protrusions, I used a fluorescent probe known to bind to activated Arf6 and the related GTPase Arf1. To address whether the ability of IpgD to stimulate exocytosis, protrusion formation, and/or Arf6/Arf1 activation requires the effector’s phosphatidylinositol-4-phosphatase activity, S. flexneri strains that express either wild type IpgD or the catalytically inactive mutant protein IpgD.C439S were used. Interestingly, the results revealed that the ability of IpgD to stimulate exocytosis, promote protrusion formation, or activate Arf6/Arf1 were independent of the effector protein’s phosphatidylinositol-4-phosphatase activity. These findings raise the intriguing possibility that IpgD controls exocytosis and protrusion formation by interacting with one or more host factors, perhaps Arf6, Arf1, or regulators of these GTPases. Collectively, the findings in this thesis provide evidence that the S. flexneri T3SS effector IpgD manipulates the human Arf proteins to induce exocytosis that promotes efficient cell-to cell spread of bacteria.