title: Cholesterol binding by the bacterial type III translocon is essential for virulence effector delivery into mammalian cells.
abstract: A ubiquitous early step in infection of man and animals by enteric bacterial pathogens like Salmonella, Shigella and enteropathogenic Escherichia coli (EPEC) is the translocation of virulence effector proteins into mammalian cells via specialized type III secretion systems (TTSSs). Translocated effectors subvert the host cytoskeleton and stimulate signalling to promote bacterial internalization or survival. Target cell plasma membrane cholesterol is central to pathogen-host cross-talk, but the precise nature of its critical contribution remains unknown. Using in vitro cholesterol-binding assays, we demonstrate that Salmonella (SipB) and Shigella (IpaB) TTSS translocon components bind cholesterol with high affinity. Direct visualization of cell-associated fluorescently labelled SipB and parallel immunogold transmission electron microscopy revealed that cholesterol levels limit both the amount and distribution of plasma membrane-integrated translocon. Correspondingly, cholesterol depletion blocked effector translocation into cultured mammalian cells by not only the related Salmonella and Shigella TTSSs, but also the more divergent EPEC system. The data reveal that cholesterol-dependent association of the bacterial TTSS translocon with the target cell plasma membrane is essential for translocon activation and effector delivery into mammalian cells.
authors.affiliation: University of Cambridge, Department of Pathology, Tennis Court Road, Cambridge CB2 1QP, UK.
authors.forename: Richard D
authors.initials: RD
authors.lastname: Hayward
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Cain
authors.forename: Emma J
authors.initials: EJ
authors.lastname: McGhie
authors.forename: Neil
authors.initials: N
authors.lastname: Phillips
authors.forename: Matthew J
authors.initials: MJ
authors.lastname: Garner
authors.forename: Vassilis
authors.initials: V
authors.lastname: Koronakis
mesh_terms: D016475:3T3 Cells / Q000382:microbiology; D000818:Animals; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism*; D002784:Cholesterol / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism / Q000472:pathogenicity; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D021962:Membrane Microdomains; D008565:Membrane Proteins / Q000378:metabolism*; D051379:Mice; D046529:Microscopy, Electron, Transmission / Q000379:methods; D021381:Protein Transport / Q000502:physiology; D012486:Salmonella typhimurium / Q000378:metabolism / Q000472:pathogenicity; D012763:Shigella flexneri / Q000378:metabolism / Q000472:pathogenicity; D014774:Virulence / Q000502:physiology
chemical_list: D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; C087863:EaeB protein, E coli; D029968:Escherichia coli Proteins; C474481:EspD protein, E coli; D008565:Membrane Proteins; C094579:invasion protein B, Salmonella typhimurium; C087447:ipaB protein, Shigella; D002784:Cholesterol