Host Complicity in Intercellular Spread of Shigella flexneri

Shigella flexneri is a Gram negative human pathogen that causes severe bacterial dysentery. Intracellular Shigella resides within the cytosol and spreads throughout the colonic epithelium. Intercellular spread occurs in the absence of an extracellular stage and is mediated by actin-based motility and the formation of bacteria-containing protrusions of the plasma membrane. Protrusions are taken up by neighboring cells and lysed by Shigella, re-initiating the infectious cycle. The mechanism of actin-based motility used by Shigella is well-studied. However, the mechanism of protrusion formation and intercellular spread remains obscure. This dissertation investigates the mechanism of intercellular spread by exploring the role played by the host proteins Dial and Dia2 and the bacterial protein IpgB2.;Intracellular Shigella adopts host Arp2/3 complex-activated actin polymerization to form actin tails that propel the bacterium through the cytosol and to the cell periphery. It was not previously known whether Shigella may use other actin assembly proteins, such as formins, during infection. Using a cell culture model of S. flexneri pathogenesis I identified the host formin proteins Dial and Dia2 as being required for efficient protrusion formation and intercellular spread. Expression of a dominant-interfering Dia construct, as well as knockdown of Dial and Dia2 protein levels, significantly reduced the efficiency of protrusion formation and dissemination by S. flexneri. The formation of actin tails and intracellular motility were unaffected. Dial localized around intracellular Shigella and in protrusions. Protrusion formation was not affected by inhibition of RhoA, a known activator of Dial and Dia2.;The bacterial type three secreted effector protein IpgB2 interacts with Dial and overexpression of IpgB2 in mammalian cells results in stress fiber formation. IpgB2 and its homolog IpgB1 are bacterial guanine nucleotide exchange factors for Rho GTPases. Using wild-type and an ipg82 mutant strain of S. flexneri I determined that IpgB2 is not required for efficient protrusion formation or intercellular spread. Instead, IpgB2 was required for efficient arrival at the plasma membrane of intracellular Shigella during the first hour of infection. In addition, I uncovered a role for RhoA in both arrival at the plasma membrane and protrusion formation that is independent of IpgB1 and IpgB2.