title: The purified Shigella IpaB and Salmonella SipB translocators share biochemical properties and membrane topology.
abstract: An essential early event in Shigella and Salmonella pathogenesis is invasion of non-phagocytic intestinal epithelial cells. Pathogen entry is triggered by the delivery of multiple bacterial effector proteins into target mammalian cells. The Shigella invasion plasmid antigen B (IpaB), which inserts into the host plasma membrane, is required for effector delivery and invasion. To investigate the biochemical properties and membrane topology of IpaB, we purified the native full-length protein following expression in laboratory Escherichia coli. Purified IpaB assembled into trimers via an N-terminal domain predicted to form a trimeric coiled-coil, and is predominantly alpha-helical. Upon lipid interaction, two transmembrane domains (residues 313-333 and 399-419) penetrate the bilayer, allowing the intervening hydrophilic region (334-398) to cross the membrane. Purified IpaB integrated into model, erythrocyte and mammalian cell membranes without disrupting bilayer integrity, and induced liposome fusion in vitro. An IpaB-derived 162 residue alpha-helical polypeptide (IpaB(418-580)) is a potent inhibitor of IpaB-directed liposome fusion in vitro and blocked Shigella entry into cultured mammalian cells at 10(-8) M. It is also a heterologous inhibitor of Salmonella invasion protein B (SipB) activity and Salmonella entry. In contrast, IpaB(418-580) failed to prevent the contact-dependent haemolytic activity of Shigella. These findings question the proposed direct link between contact-dependent haemolysis and Shigella entry, and demonstrate that IpaB and SipB share biochemical properties and membrane topology, consistent with a conserved mode of action during cell entry.
authors.affiliation: University of Cambridge, Department of Pathology, Tennis Court Road, Cambridge, CB2 1QP, UK.
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Hume
authors.forename: Emma J
authors.initials: EJ
authors.lastname: McGhie
authors.forename: Richard D
authors.initials: RD
authors.lastname: Hayward
authors.forename: Vassilis
authors.initials: V
authors.lastname: Koronakis
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism*; D002942:Circular Dichroism; D004594:Electrophysiology; D006367:HeLa Cells; D006801:Humans; D008081:Liposomes / Q000378:metabolism; D008561:Membrane Fusion; D008565:Membrane Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012475:Salmonella / Q000378:metabolism*; D012760:Shigella / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D008081:Liposomes; D008565:Membrane Proteins; D011994:Recombinant Proteins; C094579:invasion protein B, Salmonella typhimurium; C087447:ipaB protein, Shigella