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, 15 (3), 315-21

Type VI Secretion and Bacteriophage Tail Tubes Share a Common Assembly Pathway

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Type VI Secretion and Bacteriophage Tail Tubes Share a Common Assembly Pathway

Yannick R Brunet et al. EMBO Rep.

Abstract

The Type VI secretion system (T6SS) is a widespread macromolecular structure that delivers protein effectors to both eukaryotic and prokaryotic recipient cells. The current model describes the T6SS as an inverted phage tail composed of a sheath-like structure wrapped around a tube assembled by stacked Hcp hexamers. Although recent progress has been made to understand T6SS sheath assembly and dynamics, there is no evidence that Hcp forms tubes in vivo. Here we show that Hcp interacts with TssB, a component of the T6SS sheath. Using a cysteine substitution approach, we demonstrate that Hcp hexamers assemble tubes in an ordered manner with a head-to-tail stacking that are used as a scaffold for polymerization of the TssB/C sheath-like structure. Finally, we show that VgrG but not TssB/C controls the proper assembly of the Hcp tubular structure. These results highlight the conservation in the assembly mechanisms between the T6SS and the bacteriophage tail tube/sheath.

Figures

Figure 1
Figure 1
Hcp1 interacts with the sheath-like TssB1 subunit and is required for sheath assembly. A–C  Interaction between T6SS tube and sheath components. Co-immunoprecipitation assay are shown in (A) and (C). Solubilized extracts of E. coli K-12 W3110 cells producing FLAG-tagged Hcp1FL (Hcp1FL), VSV-G-tagged TssC1 (TssC1V) and HA-tagged TssB1 (TssB1HA) (A) or Hcp1FL and VSV-G-tagged TssB1 (TssB1V) (C) were subjected to immunoprecipitation with anti-FLAG-coupled beads. The input (total solubilized material, In) and the immunoprecipitated material (IP) were analyzed by 12.5%-acrylamide SDS-PAGE and proteins were immunodetected with anti-FLAG, anti-VSV-G and anti-HA monoclonal antibodies. Immunodetected proteins are indicated on the right. Molecular weight markers (in kDa) are indicated on the left. Bacterial two-hybrid assay is shown in (B). BTH101 reporter cells producing the indicated fusion were spotted on LB agar plates supplemented with X-Gal. An interaction between two fusion proteins is attested by the dark color of the colony. The TolB-T25/T18-Pal combination serves as a positive control. Non-specific interactions are ruled out using TolB-T25 or T18-Pal as negative controls. D  Time-lapse fluorescence microscopy recordings of EAEC cells producing TssB1-sfGFP. Upper panel, wild-type (WT) cells; second panel, Δhcp mutant cells; third panel, Δhcp mutant cells producing Hcp1FL (hcp+); lower panel, Δhcp mutant cells producing the S158W Hcp variant (hcpW+). Red and white arrowheads point at extensions and contractions of the T6SS sheath-like structures, respectively. Each frame is separated by 30 sec. The scale bar represents 2 μm.
Figure 2
Figure 2
Lateral cross-linking validates the Hcp1 model.

Model of the EAEC Hcp1 hexamer. Two adjacent monomers are colored in red and green, respectively. The magnification emphasizes the location of the Cys-38 (red monomer) and Asn-115 (green monomer) residues of the two adjacent monomers.

Disulfide bond formation between Hcp1 proteins within the hexamer. Cytoplasmic extracts from EAEC Δhcp1 cells producing Hcp1 or Hcp1-N115C after in vivo oxidative treatment were analyzed by 12.5%-acrylamide SDS-PAGE and proteins were immunodetected with the anti-FLAG monoclonal antibody. Positions of the Hcp1 monomer and oligomers are indicated on the right. Molecular weights (in kDa) are indicated on the left.

Figure 3
Figure 3
Hcp1 hexamers assemble tubular edifices by head-to-tail stacking in vivo. A–C  Models of EAEC Hcp1 hexamers assembled in a head-to-tail (A), tail-to-tail (B) or head-to-head (C) manner, based on the available crystal lattices. The locations of the cysteine substitutions used to probe the assembly are indicated by the yellow balls (Gly96 and Ser158 for the head-to-tail; Gln24 and Ala95 for the tail-to-tail; Gly48 for the head-to-head). D  Cytoplasmic extracts from EAEC Δhcp1 cells producing the indicated Hcp1 C38S cysteine mutant proteins after in vivo oxidative treatment were analyzed by 12.5%-acrylamide SDS-PAGE and proteins were immunodetected with the anti-FLAG monoclonal antibody. Positions of the Hcp1 monomer and oligomers are indicated on the right. Molecular weights (in kDa) are indicated on the left.
Figure 4
Figure 4
Proper assembly of Hcp1 tubes is a controlled phenomenon. A–E  Cytoplasmic extracts from EAEC Δsci-1 cells (A), ΔvgrG1 (B), ΔvgrG1 cells producing WT VgrG1 (vgrG1+) (C), Δsci-1 cells producing WT VgrG1 (Δsci-1 vgrG1+) (D), or ΔtssB1C1 cells (E) producing the cysteine-less Hcp1 variant (-) or Hcp1 variants to probe the lateral cross-linking (N115C), the head-to-tail (H-T; G96C-S158C), the tail-to-tail (T-T; Q24C-A95C), or the head-to-head (H-H; G48C) stacking modes were analyzed by 12.5%-acrylamide SDS-PAGE and proteins were immunodetected with the anti-FLAG monoclonal antibody. Position of the Hcp1 monomer is indicated on the right. Molecular weights (in kDa) are indicated on the left.

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