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Priming and polymerization of a bacterial contractile tail structure

Nature volume 531, pages 59–63 (2016)Cite this article

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Abstract

Contractile tails are composed of an inner tube wrapped by an outer sheath assembled in an extended, metastable conformation that stores mechanical energy necessary for its contraction. Contraction is used to propel the rigid inner tube towards target cells for DNA or toxin delivery. Although recent studies have revealed the structure of the contractile sheath of the type VI secretion system, the mechanisms by which its polymerization is controlled and coordinated with the assembly of the inner tube remain unknown. Here we show that the starfish-like TssA dodecameric complex interacts with tube and sheath components. Fluorescence microscopy experiments in enteroaggregative Escherichia coli reveal that TssA binds first to the type VI secretion system membrane core complex and then initiates tail polymerization. TssA remains at the tip of the growing structure and incorporates new tube and sheath blocks. On the basis of these results, we propose that TssA primes and coordinates tail tube and sheath biogenesis.

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Figure 1: In vivo imaging of sfGFP–TssA.
Figure 2: High-resolution structures of TssA domains.
Figure 3: Model of the assembly of the type VI secretion system.

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Electron Microscopy Data Bank

Protein Data Bank

Data deposits

Coordinates and structure factors have been deposited in the Protein Data Bank under accession numbers 4YO3 and 4YO5 for TssANt2 and TssACt respectively. Electron microscopy map for full-length TssA has been deposited in the Electron Microscopy Data Bank under accession code EMD-3282.

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Acknowledgements

This work was funded by the Centre National de la Recherche Scientifique, the Aix-Marseille Université, and grants from the Agence Nationale de la Recherche to E.C. (ANR-10-JCJC-1303-03), to E.C. and C.C. (ANR-14-CE14-0006-02) and from the Fondation pour la Recherche Médicale to C.C. (FRM DEQ2011-0421282) and supported by the French Infrastructure for Integrated Structural Biology (FRISBI, ANR-10-INSB-05-01). A.Z. and Y.R.B. were supported by doctoral fellowships from the French Ministry of Research. A.Z. and E.D. were supported by end-of-thesis (FDT20140931060) and post-doctoral (SPF20101221116) fellowships from the Fondation pour la Recherche Médicale, respectively. We gratefully acknowledge the Soleil synchrotron radiation facility for beamtime allocation. We thank R. Lloubès, J. Sturgis and A. Galinier for constant support, the members of the Cascales, Cambillau, Lloubès, Sturgis and Bouveret research groups for helpful discussions, E. Bouveret for providing vectors, protocols and advice for the bacterial two-hybrid assay, C. Bebeacua for preliminary electron microscopy analyses, L. Espinosa for help regarding statistical analyses, R. Lebrun and S. Lignon (proteomic platform, IMM) for mass spectrometry analyses, Y. Cully for the Supplementary Video, O. Uderso, I. Bringer and A. Brun for technical assistance, and J. D. Barras-Elatable for encouragement.

Author information

Author notes

  1. Yannick R. Brunet

    Present address: † Present address: Department of Microbiology and Immunobiology, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, Massachussets 02115, USA.,

  2. Abdelrahim Zoued and Eric Durand: These authors contributed equally to this work.

Authors and Affiliations

  1. Laboratoire d’Ingénierie des Systèmes Macromoléculaires, Institut de Microbiologie de la Méditerranée, CNRS UMR7255, Aix-Marseille Université, 31 Chemin Joseph Aiguier, Marseille Cedex 20, 13402, France

    Abdelrahim Zoued, Eric Durand, Yannick R. Brunet, Badreddine Douzi, Nicolas Flaugnatti, Laure Journet & Eric Cascales

  2. Architecture et Fonction des Macromolécules Biologiques, Centre National de la Recherche Scientifique, UMR 7257, Campus de Luminy, Case 932, Marseille Cedex 09, 13288, France

    Eric Durand, Silvia Spinelli, Badreddine Douzi & Christian Cambillau

  3. Architecture et Fonction des Macromolécules Biologiques, Aix-Marseille Université, UMR 7257, Campus de Luminy, Case 932, Marseille Cedex 09, 13288, France

    Eric Durand, Silvia Spinelli, Badreddine Douzi & Christian Cambillau

  4. G5 Biologie structurale de la sécrétion bactérienne, Institut Pasteur, 25–28 rue du Docteur Roux, Paris, 75015, France

    Eric Durand & Rémi Fronzes

  5. UMR 3528, CNRS, Institut Pasteur, 25–28 rue du Docteur Roux, Paris, 75015, France

    Eric Durand & Rémi Fronzes

  6. Laboratoire de Chimie Bactérienne, Institut de Microbiologie de la Méditerranée, CNRS UMR7283, Aix-Marseille Université, 31 Chemin Joseph Aiguier, Marseille Cedex 20, 13402, France

    Mathilde Guzzo & Tâm Mignot

  7. Synchrotron Soleil, L’Orme des merisiers, Saint-Aubin BP48, Gif-sur-Yvette Cedex, 91192, France

    Pierre Legrand

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  1. Abdelrahim Zoued
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Contributions

A.Z., E.D., C.C. and E.C. designed and conceived the experiments. C.C. and E.C supervised the execution of the experiments. A.Z., E.D., Y.R.B., S.S., B.D. and M.G. performed the experiments. A.Z. performed the in vivo experiments (BACTH, fluorescence microscopy) with the help of Y.R.B., M.G., N.F., L.J. and T.M. E.D. performed the in vitro experiments (protein purification and characterization, SAXS, electron microscopy and X-ray analyses) with the help of S.S., P.L. and R.F. B.D. performed the SPR experiments. P.L., R.F., T.M., C.C. and E.C. provided tools. E.C. wrote the paper with contributions of A.Z., E.D. and C.C.

Corresponding authors

Correspondence to Eric Durand, Christian Cambillau or Eric Cascales.

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Extended data figures and tables

Extended Data Figure 1 Hcp and TssC interact with TssA, a cytoplasmic protein required for sheath assembly and Hcp release.

a, Schematic representation of the architecture of the bacterial type VI secretion system. The scheme highlights the membrane complex anchoring the tail structure composed of the assembly baseplate, the spike, the tube and the sheath (cyto, cytoplasm; IM, inner membrane; PG, peptidoglycan layer; OM, outer membrane). b, Bacterial two-hybrid assay. BTH101 reporter cells producing the indicated proteins or domains (TssLc, cytoplasmic domain of the TssL protein; TssMc and TssMp, cytoplasmic and periplasmic domain of the TssM protein respectively) fused to the T18 or T25 domain of the Bordetella adenylate cyclase were spotted on plates supplemented with IPTG and the chromogenic substrate X-Gal. Interaction between the two fusion proteins is attested by the dark blue colour of the colony. The TolB–Pal interaction serves as a positive control. c, The absence of TssA prevents T6SS sheath dynamics. Fluorescence microscopy time-lapse recordings showing sheath dynamics using the chromosomally-encoded tssB-mCherry fusion in wild-type (WT) (tssB–mCherry pBAD33), ∆tssA (∆tssA tssB–mCherry pBAD33) and complemented ∆tssA (∆tssA tssB–mCherry pBAD33-TssAVSV-G) cells. Individual images were taken every 30 s. Assembly and contraction/disassembly events are indicated above the time-lapse images. The scale bars are 1 μm. d, The absence of TssA prevents Hcp release. Hcp release was assessed by separating whole cells (C) and supernatant (SN) fractions from 17-2 (WT), ∆tssA (∆tssA pBAD33, tssA) and complemented ∆tssA (∆tssA pBAD33-TssAVSV-G, tssAWT) cells producing Flag-epitope-tagged Hcp. A total of 1 × 108 cells and the TCA-precipitated material from the supernatant of 5 × 108 cells were analysed by western blot using anti-Flag monoclonal antibody (lower panel) and anti-TolB polyclonal antibodies as a lysis control (upper panel). The molecular weight markers (in kDa) are indicated on the left. The uncropped scans of the western blots are provided in the Supplementary Figure. e, TssA co-fractionates with cytoplasmic and membrane proteins. A fractionation procedure was applied to EAEC ∆tssA cells producing Flag-tagged TssA. Whole cells (T) were fractionated to isolate the supernatant (SN), periplasmic (P), cytoplasmic (C) and total membrane (M) fractions. Extracts from 109 (T) or 2 × 109 (SN, P, C, M) cells were separated by SDS–PAGE and immunodetected with anti-Flag monoclonal (TssA), anti-EF-Tu (cytoplasmic marker) and TolB (periplasmic marker) antibodies. The molecular weight markers (in kDa) are indicated on the left. The uncropped scans of the western blots are provided in Supplementary Figure 1.

Extended Data Figure 2 Purification and negative-stain electron microscopy analyses of the TssJLM–TssA complex.

a, TssA interacts with the TssJM complex. The total solubilized membrane extract (T) of 4 × 109 cells producing the indicated proteins was subjected to affinity chromatography using streptactin resin. Bound proteins (E) were separated by SDS–PAGE and immunodetected with anti-Flag (TssA and TssL), anti-strep tag (TssJ) and anti-5×His (TssM) monoclonal antibodies. The molecular weight markers are indicated on the left. The uncropped scans of the western blots are provided in Supplementary Figure 1. b, Superose 6 10/300 gel-filtration profile of the purified TssJLM–TssA complex. The asymmetry of the peak probably reflects the co-purification of different complexes or the dissociation of TssA from the TssJLM complex. c, Examples of representative raw particles observed for the purified TssJLM–TssA complex sample using negative-stain electron microscopy. A typical TssJLM complex is shown in red (number of particles observed n = 240) whereas a TssA-bound TssJLM complex is shown in white (n = 95). Scale bar is 10 nm. d, Magnification of the two complexes shown in c. Scale bars, 10 nm.

Extended Data Figure 3 TssA localization and dynamics.

a, Mean square displacement (MSD; in arbitrary units (a.u.)) of a representative sfGFP–TssA clusters in a wild-type strain (red line) or its ∆tssBC isogenic derivative (black line) were measured by sub-pixel tracking of fluorescent foci and plotted over time (in sec). b, Kymographic analysis reporting representative sfGFP–TssA (green) and TssB–mCherry (red) positions within the cell as a function of time. c, Representative fluorescence lifetime imaging microscopy (FLIM) of sfGFP–TssA clusters in the sfGFP–TssA/TssB–mCherry strain. A membrane-associated sfGFP–TssA cluster was chosen to define the bleached area (red circle). The laser (488 nm) was set to maximum power and focused for 3 s to ensure complete bleaching of the GFP diffusible pool. Images were taken every 30 s to follow recovery dynamics. The scale bar is 1 μm. d, Quantification of sfGFP–TssA fluorescence dynamics over time after bleaching. The dynamics of fluorescence intensity is shown over time for n = 10 independent sfGFP–TssA foci after FLIM (blue line). The fluorescence intensity of the bleached focus was also followed over time (FRAP, red line). As a control for laser focusing and intensity, membrane-associated clusters were systematically bleached in these experiments and showed no recovery suggesting the total intracellular sfGFP–TssA has been bleached by the laser. e, Representative fluorescence microscopy time-lapse recordings of the indicated ∆tssK, ∆tssE, ∆tssF, ∆tssG or ∆hcp cells producing sfGFP–TssA. Individual images were taken every 30 s. Red arrowheads indicate the localizations of TssA foci. The scale bar is 1 μm. f, Representative large fields of fluorescence microscopy analyses showing localization of sfGFP–TssA in the indicated strains. The scale bars are 1 μm. g, Box-and-whisker plots of the measured number of sfGFP–TssA foci per cell for each indicated strain. The lower and upper boundaries of the boxes correspond to the 25% and 75% percentiles respectively. The black bold horizontal bar represents the median values for each strain and the whiskers represent the 10% and 90% percentiles. Outliers are shown as open circles. A Student’s t-test was used to report significant differences (ns, not significant; ***P < 0.0001). The number of cells studied per strain (n) is indicated on top. h, Statistical analyses of sfGFP–TssA dynamics. sfGFP–TssA dynamics were categorized as ‘fixed’, ‘mobile with unidirectional trajectory’ and ‘mobile with random dynamics’ and the number of sfGFP–TssA (n, on top) foci in each category is represented as a percentage for each indicated strain. Kymographs for the two first categories are shown at the bottom of the panel. i, Schematic representation of the assembly pathway of the T6SS based on this study and available data13,15,19,24,25,55. The biogenesis starts with the initial positioning of the TssJ outer membrane lipoprotein and the sequential recruitment of the indicated subunits (from left to right). The recruitment of TssA is dependent on TssM, and that of TssK is dependent on both TssL and TssA. The exact positions of VgrG and TssE (blue) in the pathway are not known but these two subunits are not required for TssA recruitment but necessary for Hcp and TssBC polymerization.

Extended Data Figure 4 TssA interacts with tail and baseplate components.

a, TssA interaction network identified by bacterial two-hybrid analysis (see legend to Extended Data Fig. 1b). b–e, Surface plasmon resonance interaction study of TssA with its partners identified by BACTH. Sensorgrams (variation of plasmon resonance in arbitrary units (∆RU) as a function of reaction time (in sec)) were recorded upon injection of the purified native TssA protein (concentrations of 3.125 (dark grey), 6.25, 12.5, 25, 50 and 100 (light grey) μM) on HC200m chips coated with the purified N-terminal domain of VgrG (b), purified TssE (c), Hcp (d) or TssBC complex (e) (upper panels). The graph reporting ∆RU as a function of the TssA concentration (lower panel) was used to estimate the indicated apparent dissociation constants (Kd). Off-rates (percentage of dissociation 400 s after ligand injection) are indicated.

Extended Data Figure 5 TssA oligomerization and SAXS and EM structural models.

a, 10 μg of purified TssA were analysed by SDS–PAGE and Coomassie blue staining. The molecular weight markers (in kDa) are indicated on the left, and TssA and its theoretical size are indicated on the right. b, Superose 6 10/300 gel filtration profile of purified TssA (black line) and protein markers of known size (coloured lines). c, MALS/QELS/UV/RI analysis of purified TssA. The molecular mass of the TssA complex is indicated. d–j, Low-resolution SAXS model of TssA. d, Experimental scattering data calculated from an ab initio model of TssA. The square root χ value of the ‘best representative’ model is indicated. e, Representation of the Guinier plot calculated from the experimental curve. f, Pair distance distribution. g, Kratky plot representative of a multi-domain protein with flexible linkers. h–j, SAXS envelope of the ‘best representative’ model of TssA, with top (h), side (i) and tilted (j) views. The scale bar is 100 Å. k–r, Low-resolution EM model of TssA. k, Representative micrograph of the data set used for image processing. White circles indicate isolated TssA dodecamers. l. Representative selected TssA particles. m, n, Gallery of representative top (m) and side (n) class averages generated after reference-free 2D classification using Relion39. o, Fourier shell correlation (FSC) curve of the TssA reconstruction. The gold standard FSC curve was calculated in Relion39 using the masked reconstruction of TssA. p–r, Top (p), side (q) and tilted (r) views of the three-dimensional reconstruction model of the TssA dodecamer obtained by electron microscopy (accession number: EMD-3282). The scale bar is 50 Å. Whereas the SAXS model allows to better visualize the arm length compared to the EM reconstruction, its low resolution impairs the visual separation of the dimeric arms.

Extended Data Figure 6 Identification, oligomerization and interaction analysis of TssA domains.

a, Limited proteolysis of purified TssA. The purified full-length TssA protein (first lane) was submitted to proteinase K limited proteolysis for the time indicated on top of each lane and analysed by SDS–PAGE and Coomassie blue staining. Stable fragments are indicated on the right with their boundaries (numbers identified in the sequence in b) and the corresponding fragment. The uncropped scan of the Coomassie blue stained gel is provided in Supplementary Figure 1. b, TssA protein sequence. The localization of the boundaries of the stable fragments obtained after Proteinase K limited proteolysis and electrospray mass spectrometry analyses are arrowed. The secondary structures observed in the crystal structures (Fig. 2a and Extended Data Fig. 6f, g) are indicated on top of the corresponding sequence. c, Bacterial two-hybrid analysis of TssANt and TssACt interactions (see legend to Extended Data Fig. 1b). d, e, MALS/QELS/UV/RI analysis of the purified TssANt (d) and TssACt (e) fragments. f, g, X-ray structure of the TssANt2 domain (PDB: 4YO3). The rainbow coloured ribbon representation of the TssANt monomer is shown (f, consecutive α-helices numbered α1 to α7) whereas the dimeric structure (g) highlights the helices at the interface (α1, α2 and α6). h, The TssA central core interacts with Hcp and VgrG whereas the TssA arms interact with TssE and TssC. Bacterial two-hybrid analysis of TssANt and TssACt interactions (see legend to Extended Data Fig. 1b). i, j, Surface plasmon resonance interaction study of the purified TssACt (i) or TssANt (j) domains with the Hcp protein (i) or the TssBC complex (j). Sensorgrams (variation of plasmon resonance in arbitrary unit (∆RU) as a function of reaction time (in seconds)) were recorded upon injection of the purified TssA C-terminal (i) or TssANt (j) domains (concentrations of 3.125 (dark grey), 6.25, 12.5, 25, 50 and 100 (light grey) μM) on HC200m chips coated with the purified Hcp protein (i) or the TssBC complex (j) (upper panels). The graph reporting ∆RU as a function of the TssA domain concentration (lower panel) was used to estimate the indicated apparent dissociation constants (Kd).

Extended Data Figure 7 Comparison of the SAXS, EM and X-ray structures of TssA.

a, Schematic representation and colour code of the constructs used for SAXS (grey), electron microscopy (light blue) and X-ray (TssANt, dark blue; TssACt, red) analyses. The epitopes and theoretical molecular masses of the domains are indicated. TRX, thioredoxine; N, N terminus; C, C terminus. b, Fit between the experimental data (green dots) and the calculated scattering curves for TssANt2 and TssACt generated by CRYSOL (red line). c–f, SAXS/X-ray comparison. Top (c), side (d) and bottom (f) views of the fitting of TssANt2 (blue ribbon) and TssACt (red ribbon) X-ray structures into the TssA SAXS envelope (transparent grey surface). Scale bars are 10 nm. e, Magnification of a cut-away section of the fitting shown in d. Scale bar is 5 nm. g–i, SAXS/EM/X-ray comparison. Top (g) and side (h) views of the superimposition of SAXS (grey surface), EM (transparent light-blue surface) and X-ray structures of TssA. Scale bars are 10 nm. i, Magnification of a cut-away section of the superimposition shown in h. j–n, EM/X-ray comparison. Top (j), side (k) and bottom (l) views of the fitting of TssANt2 (blue ribbon) and TssACt (red ribbon) X-ray structures into the TssA EM envelope (transparent grey surface). Scale bars are 10 nm. m, n, Magnifications of the top and bottom views of the docking of the TssA domain X-ray structures into the TssA EM map highlighting the interface between the TssA central core (TssACt, red ribbon) and arms (TssANt2, blue ribbon). The C-terminal helix of TssANt2 (ends at position 377) and N-terminal helix of TssACt (starts at position 395) are shown in yellow. o, Top view of the fitting of the X-ray structure of EAEC Hcp (green ribbon, PDB 4HKH56) into the TssA SAXS envelope (grey surface). The scale bar is 10 nm.

Extended Data Figure 8 Models of tail-sheath TssA complexes and comparison between the bacteriophage T4 gp15 and T6SS TssA subunits.

a, b, Surface (a) and cross-section (b) views of the complex of TssA (EM map, blue) with the extended tail sheath model (the last four rows shown in different colours). In the cut-away section, four stacks of Hcp rings are visible. As shown by bacterial two-hybrid and SPR analyses, Hcp contacts the TssACt central core whereas TssBC contacts the TssANt arms. The TssA arms fit between the TssBC monomers of the last row. c, Surface view of the complex of TssA (EM map, blue) with the contracted tail sheath model (the last four rows shown in different colours), highlighting the loose packing between TssA and the tail sheath in this conformation, suggesting that TssA might dissociate after sheath contraction. d–f, Comparison between the bacteriophage T4 gp15 and T6SS TssA subunits. d, e, Schematic representations of the bacteriophage T4 tail distal end comprising the gp19 tube (grey) and gp18 sheath (blue) proteins and the gp3 (green) and gp15 (red) neck proteins (d) and the T6SS tail distal end comprising the Hcp tube (grey) and TssBC sheath (blue) proteins and the TssA dodecamer (red) (e). The possibility that a functional homologue of bacteriophage T4 gp3 exists is shown by the question mark. f, Fitting of the model of the gp15 structure in complex with the last row of the gp18 sheath (in purple)29 in the TssA SAXS envelope (grey surface).

Extended Data Table 1 SAXS parameters of TRX–TssA
Full size table
Extended Data Table 2 Data collection, phasing and refinement statistics for SAD (SeMet) structures
Full size table

Supplementary information

Supplementary Table

This file shows the strains, plasmids and oligonucleotides used in this study. (PDF 294 kb)

Supplementary Figure

Uncropped scans of western blots and gels presented in Extended Data Fig. 1d, 1e, 2a & 6a. Molecular weight markers (in kDa) are indicated. (PDF 617 kb)

Dynamic representation of T6SS assembly highlighting the TssA mode of action

The TssA protein (red ring) bound to the membrane and baseplate complexes (TssJLM shown in yellow/orange; VgrG spike, purple; baseplate, orange) initiates tail polymerization. Polymerization is pursued by the TssA-dependent sequential and processive recruitement of Hcp rings (green rings) and TssBC strands (blue rings). Once assembled, the sheath contracts and propels the Hcp/VgrG needle to the cell exterior. (MOV 1732 kb)

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Zoued, A., Durand, E., Brunet, Y. et al. Priming and polymerization of a bacterial contractile tail structure. Nature 531, 59–63 (2016). https://doi.org/10.1038/nature17182

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