title: Salmonella-directed recruitment of new membrane to invasion foci via the host exocyst complex.
abstract: Salmonella attachment to the intestinal epithelium triggers delivery of bacterial effector proteins into the host cytosol through a type III secretion system (T3SS), leading to pronounced membrane ruffling and macropinocytic uptake of attached bacteria. The tip of the T3SS is made up of two proteins, SipB and SipC, which insert into the host plasma membrane, forming a translocation pore. Both the N and C termini of SipC are exposed in the host cytosol and have been shown to directly modulate actin cytoskeleton assembly. We have identified a direct interaction between SipC and Exo70, a component of the exocyst complex, which mediates docking and fusion of exocytic vesicles with the plasma membrane. Here, we show that exocyst components coprecipitate with SipC and accumulate at sites of invasion by Salmonella typhimurium. Exocyst assembly requires activation of the small GTPase RalA, which we show is triggered during Salmonella infection by the translocated effector, SopE. Knockdown of RalA or Sec5 results in reduced membrane ruffling at sites of attachment and impairs bacterial entry into host cells. These findings suggest that S. typhimurium enhances invasion efficiency by promoting localized membrane expansion, directly through SipC-dependent recruitment of the exocyst and indirectly via SopE-dependent activation of RalA.
authors.affiliation: Department of Microbiology, University of Virginia Health System, Charlottesville, VA 22908-0732, USA.
authors.forename: Christina D
authors.initials: CD
authors.lastname: Nichols
authors.forename: James E
authors.initials: JE
authors.lastname: Casanova
mesh_terms: D001426:Bacterial Proteins / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism*; D002846:Chromatography, Affinity; D005089:Exocytosis / Q000502:physiology*; D005434:Flow Cytometry; D006367:HeLa Cells; D006801:Humans; D008856:Microscopy, Fluorescence; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics; D012480:Salmonella Infections / Q000378:metabolism*; D012486:Salmonella typhimurium / Q000378:metabolism*; D055571:Secretory Pathway / Q000502:physiology*; D020798:Two-Hybrid System Techniques; D033921:Vesicular Transport Proteins / Q000378:metabolism; D020725:ral GTP-Binding Proteins / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; C470182:EXOC2 protein, human; C547754:EXOC7 protein, human; D034741:RNA, Small Interfering; C094578:Salmonella invasion protein C; C103987:SopE protein, Salmonella; D033921:Vesicular Transport Proteins; C492198:RALA protein, human; D020725:ral GTP-Binding Proteins