title: Rho family GTPase Cdc42 is essential for the actin-based motility of Shigella in mammalian cells.
abstract: Shigella, the causative agent of bacillary dysentery, is capable of directing its movement within host cells by exploiting actin dynamics. The VirG protein expressed at one pole of the bacterium can recruit neural Wiskott-Aldrich syndrome protein (N-WASP), a downstream effector of Cdc42. Here, we show that Cdc42 is required for the actin-based motility of Shigella. Microinjection of a dominant active mutant Cdc42, but not Rac1 or RhoA, into Swiss 3T3 cells accelerated Shigella motility. In add-back experiments in Xenopus egg extracts, addition of a guanine nucleotide dissociation inhibitor for the Rho family, RhoGDI, greatly diminished the bacterial motility or actin assembly, which was restored by adding activated Cdc42. In N-WASP-depleted extracts, the bacterial movement almost arrested was restored by adding exogenous N-WASP but not H208D, an N-WASP mutant defective in binding to Cdc42. In pyrene actin assay, Cdc42 enhanced VirG-stimulating actin polymerization by N-WASP-actin-related protein (Arp)2/3 complex. Actually, Cdc42 stimulated actin cloud formation on the surface of bacteria expressing VirG in a solution containing N-WASP, Arp2/3 complex, and G-actin. Immunohistological study of Shigella-infected cells expressing green fluorescent protein-tagged Cdc42 revealed that Cdc42 accumulated by being colocalized with actin cloud at one pole of intracellular bacterium. Furthermore, overexpression of H208D mutant in cells interfered with the actin assembly of infected Shigella and diminished the intra- and intercellular spreading. These results suggest that Cdc42 activity is involved in initiating actin nucleation mediated by VirG-N-WASP-Arp2/3 complex formed on intracellular Shigella.
authors.affiliation: Department of Bacteriology, Institute of Medical Science, University of Tokyo, Japan.
authors.forename: T
authors.initials: T
authors.lastname: Suzuki
authors.forename: H
authors.initials: H
authors.lastname: Mimuro
authors.forename: H
authors.initials: H
authors.lastname: Miki
authors.forename: T
authors.initials: T
authors.lastname: Takenawa
authors.forename: T
authors.initials: T
authors.lastname: Sasaki
authors.forename: H
authors.initials: H
authors.lastname: Nakanishi
authors.forename: Y
authors.initials: Y
authors.lastname: Takai
authors.forename: C
authors.initials: C
authors.lastname: Sasakawa
mesh_terms: D016475:3T3 Cells; D000199:Actins / Q000378:metabolism*; D000818:Animals; D001426:Bacterial Proteins / Q000378:metabolism; D019556:COS Cells; D004268:DNA-Binding Proteins / Q000378:metabolism; D004285:Dogs; D020730:Guanine Nucleotide Dissociation Inhibitors; D006801:Humans; D008322:Mammals; D051379:Mice; D008845:Microinjections / Q000379:methods; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D010063:Ovum / Q000378:metabolism; D011817:Rabbits; D051381:Rats; D012763:Shigella flexneri / Q000502:physiology*; D014157:Transcription Factors / Q000378:metabolism; D051317:Wiskott-Aldrich Syndrome Protein, Neuronal; D014981:Xenopus / Q000378:metabolism; D020764:cdc42 GTP-Binding Protein / Q000378:metabolism*; D062948:rho Guanine Nucleotide Dissociation Inhibitor alpha; D062947:rho-Specific Guanine Nucleotide Dissociation Inhibitors
chemical_list: C569638:ARHGDIA protein, human; D000199:Actins; D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D020730:Guanine Nucleotide Dissociation Inhibitors; D009419:Nerve Tissue Proteins; D014157:Transcription Factors; C495771:WASL protein, human; C495772:Wasl protein, mouse; C495770:Wasl protein, rat; D051317:Wiskott-Aldrich Syndrome Protein, Neuronal; D062948:rho Guanine Nucleotide Dissociation Inhibitor alpha; D062947:rho-Specific Guanine Nucleotide Dissociation Inhibitors; C058483:virG protein, Shigella flexneri; D020764:cdc42 GTP-Binding Protein