title: Molecular dissection of Salmonella-induced membrane ruffling versus invasion.
abstract: Type III secretion system-mediated injection of a cocktail of bacterial proteins drives actin rearrangements, frequently adopting the shape of prominent protuberances of ruffling membrane, and culminating in host cell invasion of Gram-negative pathogens like Salmonella typhimurium. Different Salmonella effectors are able to bind actin and activate Rho-family GTPases, which have previously been implicated in mediating actin-dependent Salmonella entry by interacting with N-WASP or WAVE-complex, well-established activators of the actin nucleation machine Arp2/3-complex. Using genetic deletion and RNA interference studies, we show here that neither individual nor collective removal of these Arp2/3- complex activators affected host cell invasion as efficiently as Arp2/3-complex knock-down, although the latter was also not essential. However, interference with WAVE-complex function abrogated Salmonella-induced membrane ruffling without significantly affecting entry efficiency, actin or Arp2/3-complex accumulation. In addition, scanning electron microscopy images captured entry events in the absence of prominent membrane ruffles. Finally, localization and RNA interference studies indicated a relevant function in Salmonella entry for the novel Arp2/3-complex regulator WASH. These data establish for the first time that Salmonella invasion is separable from bacteria-induced membrane ruffling, and uncover an additional Arp2/3-complex activator as well as an Arp2/3-complex-independent actin assembly activity that contribute to Salmonella invasion.
authors.affiliation: Helmholtz Centre for Infection Research, Braunschweig, Germany.
authors.forename: Jan
authors.initials: J
authors.lastname: Hänisch
authors.forename: Julia
authors.initials: J
authors.lastname: Ehinger
authors.forename: Markus
authors.initials: M
authors.lastname: Ladwein
authors.forename: Manfred
authors.initials: M
authors.lastname: Rohde
authors.forename: Emmanuel
authors.initials: E
authors.lastname: Derivery
authors.forename: Tanja
authors.initials: T
authors.lastname: Bosse
authors.forename: Anika
authors.initials: A
authors.lastname: Steffen
authors.forename: Dirk
authors.initials: D
authors.lastname: Bumann
authors.forename: Benjamin
authors.initials: B
authors.lastname: Misselwitz
authors.forename: Wolf-Dietrich
authors.initials: WD
authors.lastname: Hardt
authors.forename: Alexis
authors.initials: A
authors.lastname: Gautreau
authors.forename: Theresia E B
authors.initials: TE
authors.lastname: Stradal
authors.forename: Klemens
authors.initials: K
authors.lastname: Rottner
mesh_terms: D051376:Actin-Related Protein 2-3 Complex; D001426:Bacterial Proteins / Q000235:genetics / Q000502:physiology*; D015153:Blotting, Western; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism* / Q000382:microbiology / Q000648:ultrastructure; D005347:Fibroblasts / Q000378:metabolism / Q000382:microbiology / Q000648:ultrastructure; D006801:Humans; D008855:Microscopy, Electron, Scanning; D008856:Microscopy, Fluorescence; D011506:Proteins / Q000235:genetics / Q000502:physiology; D034622:RNA Interference; D012486:Salmonella typhimurium / Q000502:physiology*; D051317:Wiskott-Aldrich Syndrome Protein, Neuronal / Q000378:metabolism / Q000502:physiology; D012359:tRNA Methyltransferases
chemical_list: D051376:Actin-Related Protein 2-3 Complex; D001426:Bacterial Proteins; D011506:Proteins; C495771:WASL protein, human; D051317:Wiskott-Aldrich Syndrome Protein, Neuronal; C498548:TRMO protein, human; D012359:tRNA Methyltransferases