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 apparatus are essential for reorganization of the cytoskeleton during cell invasion. We have developed a semi-permeabilized cell assay that tests the effects of bacterial proteins on the actin cytoskeleton. The Shigella IpaC protein was found to induce the formation of filopodial and lamellipodial extensions in these semi-permeabilized cells. Microinjection of IpaC into cells, 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 IpaC mAb stimulated extensive lamellae formation. Shigella induced foci of actin polymerization in the permeabilized cells and these were inhibited by anti-C-terminal IpaC mAbs. Consistent with a role for IpaC in Shigella-induced 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 Cdc42 or the Cdc42-binding domain of WASP, whereas a dominant-interfering form of Rac resulted in inhibition of lamellae formation. We conclude that IpaC leads to activation of Cdc42 which in turn, causes activation of Rac, both GTPases being required for Shigella entry.
Tran Van Nhieu
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Unité de Pathogénie Microbienne Moléculaire INSERM U389, Institut Pasteur, 28 rue du Dr Roux, 75724 Paris Cedex 15, France. gtranvan@pasteur.fr
Caron
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Hall
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Sansonetti
P J
PJ
D016475:3T3 Cells; D000199:Actins / Q000378:metabolism*; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000494:pharmacology; D000942:Antigens, Bacterial / Q000008:administration & dosage / Q000235:genetics / Q000276:immunology / Q000502:physiology*; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism; D002463:Cell Membrane Permeability; D003599:Cytoskeleton / Q000187:drug effects / Q000378:metabolism; D004705:Endocytosis / Q000187:drug effects; D004847:Epithelial Cells / Q000187:drug effects / Q000201:enzymology / Q000382:microbiology* / Q000648:ultrastructure*; D020558:GTP Phosphohydrolases / Q000235:genetics / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism; D005799:Genes, Dominant / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D009154:Mutation; D011108:Polymers; D011506:Proteins / Q000235:genetics / Q000378:metabolism; D011554:Pseudopodia / Q000187:drug effects / Q000378:metabolism*; D012760:Shigella / Q000378:metabolism / Q000472:pathogenicity / Q000502:physiology*; D014162:Transfection; D051316:Wiskott-Aldrich Syndrome Protein; D020764:cdc42 GTP-Binding Protein; D020744:rac GTP-Binding Proteins
D000199:Actins; D000911:Antibodies, Monoclonal; D000942:Antigens, Bacterial; D018797:Cell Cycle Proteins; D011108:Polymers; D011506:Proteins; C495767:WAS protein, human; C495768:Was protein, mouse; D051316:Wiskott-Aldrich Syndrome Protein; C092342:IpaC protein, Shigella; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; D020764:cdc42 GTP-Binding Protein; D020744:rac GTP-Binding Proteins