IpaC induces actin polymerization and filopodia formation during Shigella entry into epithelial cells

Shigella proteins that are targeted to host cells by a type III secretion a pparatus are essential for reorganization of the cytoskeleton during cell i nvasion. We have developed a semi-permeabilized cell assay that tests the e ffects of bacterial proteins on the actin cytoskeleton, The Shigella IpaC p rotein was found to induce the formation of filopodial and lamellipodial ex tensions in these semi-permeabilized cells. Microinjection of IpaC into cel ls, or cellular expression of IpaC also led to the formation of filopodial structures. Monoclonal antibodies (mAbs) directed against the C-terminus of IpaC inhibited the IpaC-induced extensions, whereas an anti-N-terminal Ipa C mAb stimulated extensive lamellae formation, Shigella induced foci of act in polymerization in the permeabilized cells and these were inhibited by an ti-C-terminal IpaC mAbs. Consistent with a role for IpaC in Shigella-induce d cytoskeletal rearrangements during entry, stable transfectants expressing IpaC challenged with Shigella showed increased bacterial internalization. IpaC-induced extensions were inhibited by a dominant-interfering form of Cd c42 or the Cdc42-binding domain of WASP, whereas a dominant-interfering for m of Rac resulted in inhibition of lamellae formation, We conclude that Ipa C leads to activation of Cdc42 which in turn, causes activation of Rac, bot h GTPases being required for Shigella entry,