title: The common structural architecture of Shigella flexneri and Salmonella typhimurium type three secretion needles.
abstract: The Type Three Secretion System (T3SS), or injectisome, is a macromolecular infection machinery present in many pathogenic Gram-negative bacteria. It consists of a basal body, anchored in both bacterial membranes, and a hollow needle through which effector proteins are delivered into the target host cell. Two different architectures of the T3SS needle have been previously proposed. First, an atomic model of the Salmonella typhimurium needle was generated from solid-state NMR data. The needle subunit protein, PrgI, comprises a rigid-extended N-terminal segment and a helix-loop-helix motif with the N-terminus located on the outside face of the needle. Second, a model of the Shigella flexneri needle was generated from a high-resolution 7.7-Å cryo-electron microscopy density map. The subunit protein, MxiH, contains an N-terminal α-helix, a loop, another α-helix, a 14-residue-long β-hairpin (Q51-Q64) and a C-terminal α-helix, with the N-terminus facing inward to the lumen of the needle. In the current study, we carried out solid-state NMR measurements of wild-type Shigella flexneri needles polymerized in vitro and identified the following secondary structure elements for MxiH: a rigid-extended N-terminal segment (S2-T11), an α-helix (L12-A38), a loop (E39-P44) and a C-terminal α-helix (Q45-R83). Using immunogold labeling in vitro and in vivo on functional needles, we located the N-terminus of MxiH subunits on the exterior of the assembly, consistent with evolutionary sequence conservation patterns and mutagenesis data. We generated a homology model of Shigella flexneri needles compatible with both experimental data: the MxiH solid-state NMR chemical shifts and the state-of-the-art cryoEM density map. These results corroborate the solid-state NMR structure previously solved for Salmonella typhimurium PrgI needles and establish that Shigella flexneri and Salmonella typhimurium subunit proteins adopt a conserved structure and orientation in their assembled state. Our study reveals a common structural architecture of T3SS needles, essential to understand T3SS-mediated infection and develop treatments.
authors.affiliation: Department of NMR-based Structural Biology, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany.
authors.forename: Jean-Philippe
authors.initials: JP
authors.lastname: Demers
authors.forename: Nikolaos G
authors.initials: NG
authors.lastname: Sgourakis
authors.forename: Rashmi
authors.initials: R
authors.lastname: Gupta
authors.forename: Antoine
authors.initials: A
authors.lastname: Loquet
authors.forename: Karin
authors.initials: K
authors.lastname: Giller
authors.forename: Dietmar
authors.initials: D
authors.lastname: Riedel
authors.forename: Britta
authors.initials: B
authors.lastname: Laube
authors.forename: Michael
authors.initials: M
authors.lastname: Kolbe
authors.forename: David
authors.initials: D
authors.lastname: Baker
authors.forename: Stefan
authors.initials: S
authors.lastname: Becker
authors.forename: Adam
authors.initials: A
authors.lastname: Lange
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000648:ultrastructure; D020285:Cryoelectron Microscopy; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012486:Salmonella typhimurium / Q000737:chemistry* / Q000648:ultrastructure; D016415:Sequence Alignment; D012763:Shigella flexneri / Q000737:chemistry* / Q000648:ultrastructure
chemical_list: D001426:Bacterial Proteins; C413572:MxiH protein, Shigella
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