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Atomic structures of an entire contractile injection system in both the extended and contracted states

Abstract

Contractile injection systems are sophisticated multiprotein nanomachines that puncture target cell membranes. Although the number of atomic-resolution insights into contractile bacteriophage tails, bacterial type six secretion systems and R-pyocins is rapidly increasing, structural information on the contraction of bacterial phage-like protein-translocation structures directed towards eukaryotic hosts is scarce. Here, we characterize the antifeeding prophage AFP from Serratia entomophila by cryo-electron microscopy. We present the high-resolution structure of the entire AFP particle in the extended state, trace 11 protein chains de novo from the apical cap to the needle tip, describe localization variants and perform specific structural comparisons with related systems. We analyse inter-subunit interactions and highlight their universal conservation within contractile injection systems while revealing the specificities of AFP. Furthermore, we provide the structure of the AFP sheath–baseplate complex in a contracted state. This study reveals atomic details of interaction networks that accompany and define the contraction mechanism of toxin-delivery tailocins, offering a comprehensive framework for understanding their mode of action and for their possible adaptation as biocontrol agents.

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Fig. 1: Cryo-EM structures of the complete AFP contractile injection system in the extended and contracted states.
Fig. 2: Molecular organization of the inner tube of AFP in the extended state.
Fig. 3: Molecular organization of the AFP sheath in the extended and contracted states.
Fig. 4: Architecture of the apical cap of AFP in the extended state.
Fig. 5: Molecular organization of the AFP baseplate in the extended and contracted states.

Data availability

The cryo-EM maps and corresponding atomic coordinates have been deposited to the EMDB and PDB with the following accession codes: full extended AFP composite map, EMD-4783; extended AFP baseplate C6 map, EMD-4782 and 6RAO; extended AFP baseplate C3 map, EMD-4800 and 6RBK; extended AFP cap ending in Afp2–Afp16 EMD-4784 and 6RAP; extended AFP cap ending in Afp3–Afp16, EMD-4801; extended AFP sheath map, EMD-4802 and 6RBN; extended AFP needle map from subtracted images, EMD-4871; contracted AFP sheath map, EMD-4803 and 6RC8; contracted AFP baseplate; EMD-4876 and 6RGL and contracted AFP sheath C6 map, EMD-4859. All other data supporting the findings of this study are available from A.D. and I.G. on request.

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Acknowledgements

This work was supported by a RSNZ Marsden grant to A.K.M. and M.R.H.H. and by the Bio-protection Research Centre. The I.G. lab was funded by a European Union’s Horizon 2020 research and innovation programme under grant agreement no. 647784. A.D. was further supported by the Fondation Recherche Medicale (grant no. ARF20160936266) and Labex GRAL (grant no. ANR-10-LABX-49-01). J.F. was supported by a long-term EMBO fellowship (grant no. ALTF441-2017). M.J. was supported by a doctoral grant from the Commissariat à l’Énergie Atomique et aux Énergies Alternatives (CEA). The authors wish to acknowledge the New Zealand eScience Infrastructure (NeSI) high performance computing facilities—particularly B. Roberts and P. Maxwell who set up the cryo-EM software on the cluster and provided precious support—for the image processing. The national facilities of New Zealand are provided by the NeSI and are jointly funded by collaborator institutions of the NeSI and through the Research Infrastructure programme of the Ministry of Business, Innovation and Employment (https://www.nesi.org.nz). We also acknowledge A. Peuch and G. Schoehn for support and access to the joint IBS/EMBL EM computing cluster, which was used as part of the platforms of the Grenoble Instruct-ERIC center (ISBG; UMS 3518 CNRS-CEA-UGA-EMBL) within the Grenoble Partnership for Structural Biology. Platform access was supported by FRISBI (ANR-10-INBS-05-02) and GRAL, a project of the University Grenoble Alpes graduate school (Ecoles Universitaires de Recherche) CBH-EUR-GS (ANR-17-EURE-0003). IBS acknowledges integration into the Interdisciplinary Research Institute of Grenoble (IRIG, CEA). We thank A. Jakobi for advice and help with the atomic model refinements and local amplitude scaling of the maps, and for providing early versions of LocScale. We thank A. Turner and the Imaging Centre of the University of Auckland for support and help with the use of the electron microscopes. We thank C. Sachse for help and advice in image processing and in setting SPRING on the NeSI and the IBS/EMBL clusters. We thank M. Middleditch for the mass-spectrometry experiments and help with the data analysis. The EMBL Cryo-Electron Microscopy Service Platform is acknowledged for support in image acquisition and analysis. We are grateful to W. Hagen for the acquisition of high-quality cryo-EM images of extended AFP in 2016. Data acquisition has been supported by iNEXT (grant no. 653706) funded by the EU Horizon 2020 programme. We acknowledge P. Leiman for critical comments of the manuscript.

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A.K.M. and M.R.H.H. designed and funded the project. A.D., H.V. and M.J. purified and initially characterized samples by negative staining and cryo-EM. A.D. analysed the mass-spectrometry data. A.D. performed the image analyses with significant input from H.V. A.D. performed the atomic model building with significant input from H.V. and J.F. J.H. and H.J. collected the high-resolution cryo-EM datasets of the contracted AFP. A.D., H.V., T.J., J.F., M.J., J.B.H., M.R.H.H., I.G. and A.K.M. analysed the data. A.D., J.F., M.J. and I.G. prepared the figures and tables. A.K.M., M.R.H.H. and I.G. provided support and supervised the project at various stages. I.G. and A.K.M. wrote the manuscript with significant input from M.R.H.H., J.B.H., A.D., J.F. and M.J., and contributions from all of the authors.

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Correspondence to Mark R. H. Hurst or Irina Gutsche or Alok K. Mitra.

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Desfosses, A., Venugopal, H., Joshi, T. et al. Atomic structures of an entire contractile injection system in both the extended and contracted states. Nat Microbiol 4, 1885–1894 (2019). https://doi.org/10.1038/s41564-019-0530-6

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