title: Three-dimensional electron microscopy reconstruction and cysteine-mediated crosslinking provide a model of the type III secretion system needle tip complex.
abstract: Type III secretion systems are found in many Gram-negative bacteria. They are activated by contact with eukaryotic cells and inject virulence proteins inside them. Host cell detection requires a protein complex located at the tip of the device's external injection needle. The Shigella tip complex (TC) is composed of IpaD, a hydrophilic protein, and IpaB, a hydrophobic protein, which later forms part of the injection pore in the host membrane. Here we used labelling and crosslinking methods to show that TCs from a ΔipaB strain contain five IpaD subunits while the TCs from wild-type can also contain one IpaB and four IpaD subunits. Electron microscopy followed by single particle and helical image analysis was used to reconstruct three-dimensional images of TCs at ∼ 20 Å resolution. Docking of an IpaD crystal structure, constrained by the crosslinks observed, reveals that TC organisation is different from that of all previously proposed models. Our findings suggest new mechanisms for TC assembly and function. The TC is the only site within these secretion systems targeted by disease-protecting antibodies. By suggesting how these act, our work will allow improvement of prophylactic and therapeutic strategies.
authors.affiliation: Schools of Cellular & Molecular Medicine and Biochemistry, University of Bristol, Bristol, BS8 1TD, UK.
authors.forename: Martin
authors.initials: M
authors.lastname: Cheung
authors.forename: Da-Kang
authors.initials: DK
authors.lastname: Shen
authors.forename: Fumiaki
authors.initials: F
authors.lastname: Makino
authors.forename: Takayuki
authors.initials: T
authors.lastname: Kato
authors.forename: A Dorothea
authors.initials: AD
authors.lastname: Roehrich
authors.forename: Isabel
authors.initials: I
authors.lastname: Martinez-Argudo
authors.forename: Matthew L
authors.initials: ML
authors.lastname: Walker
authors.forename: Isabel
authors.initials: I
authors.lastname: Murillo
authors.forename: Xia
authors.initials: X
authors.lastname: Liu
authors.forename: Maria
authors.initials: M
authors.lastname: Pain
authors.forename: James
authors.initials: J
authors.lastname: Brown
authors.forename: Gordon
authors.initials: G
authors.lastname: Frazer
authors.forename: Judith
authors.initials: J
authors.lastname: Mantell
authors.forename: Petros
authors.initials: P
authors.lastname: Mina
authors.forename: Thomas
authors.initials: T
authors.lastname: Todd
authors.forename: Richard B
authors.initials: RB
authors.lastname: Sessions
authors.forename: Keiichi
authors.initials: K
authors.lastname: Namba
authors.forename: Ariel J
authors.initials: AJ
authors.lastname: Blocker
mesh_terms: D000942:Antigens, Bacterial / Q000737:chemistry* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D058947:Bacterial Secretion Systems; D003432:Cross-Linking Reagents / Q000378:metabolism; D003545:Cysteine / Q000378:metabolism*; D021621:Imaging, Three-Dimensional; D008854:Microscopy, Electron; D008958:Models, Molecular; D062105:Molecular Docking Simulation; D055503:Protein Multimerization; D017433:Protein Structure, Secondary; D012763:Shigella flexneri / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D058947:Bacterial Secretion Systems; D003432:Cross-Linking Reagents; C095770:IpaD protein, Shigella flexneri; C087447:ipaB protein, Shigella; D003545:Cysteine