title: The target cell plasma membrane is a critical interface for Salmonella cell entry effector-host interplay.
abstract: Salmonella species trigger host membrane ruffling to force their internalization into non-phagocytic intestinal epithelial cells. This requires bacterial effector protein delivery into the target cell via a type III secretion system. Six translocated effectors manipulate cellular actin dynamics, but how their direct and indirect activities are spatially and temporally co-ordinated to promote productive cytoskeletal rearrangements remains essentially unexplored. To gain further insight into this process, we applied mechanical cell fractionation and immunofluorescence microscopy to systematically investigate the subcellular localization of epitope-tagged effectors in transiently transfected and Salmonella-infected cultured cells. Although five effectors contain no apparent membrane-targeting domains, all six localized exclusively in the target cell plasma membrane fraction and correspondingly were visualized at the cell periphery, from where they induced distinct effects on the actin cytoskeleton. Unexpectedly, no translocated effector pool was detectable in the cell cytosol. Using parallel in vitro assays, we demonstrate that the prenylated cellular GTPase Cdc42 is necessary and sufficient for membrane association of the Salmonella GTP exchange factor and GTPase-activating protein mimics SopE and SptP, which have no intrinsic lipid affinity. The data show that the host plasma membrane is a critical interface for effector-target interaction, and establish versatile systems to further dissect effector interplay.
authors.affiliation: University of Cambridge, Department of Pathology, Tennis Court Road, Cambridge CB2 1QP, UK.
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Cain
authors.forename: Richard D
authors.initials: RD
authors.lastname: Hayward
authors.forename: Vassilis
authors.initials: V
authors.lastname: Koronakis
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000378:metabolism*; D002458:Cell Fractionation; D002462:Cell Membrane / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism; D004847:Epithelial Cells / Q000166:cytology / Q000382:microbiology; D007150:Immunohistochemistry; D007413:Intestinal Mucosa / Q000166:cytology / Q000382:microbiology; D051379:Mice; D041681:NIH 3T3 Cells; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012486:Salmonella typhimurium / Q000166:cytology / Q000378:metabolism* / Q000472:pathogenicity*; D015398:Signal Transduction / Q000502:physiology; D020764:cdc42 GTP-Binding Protein / Q000737:chemistry / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins; C103987:SopE protein, Salmonella; D020764:cdc42 GTP-Binding Protein