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Near-atomic-resolution cryo-EM analysis of the Salmonella T3S injectisome basal body

Nature volume 540, pages 597601 (22 December 2016) | Download Citation

Abstract

The type III secretion (T3S) injectisome is a specialized protein nanomachine that is critical for the pathogenicity of many Gram-negative bacteria, including purveyors of plague, typhoid fever, whooping cough, sexually transmitted infections and major nosocomial infections. This syringe-shaped 3.5-MDa macromolecular assembly spans both bacterial membranes and that of the infected host cell. The internal channel formed by the injectisome allows for the direct delivery of partially unfolded virulence effectors into the host cytoplasm1. The structural foundation of the injectisome is the basal body, a molecular lock-nut structure composed predominantly of three proteins that form highly oligomerized concentric rings spanning the inner and outer membranes2,3,4,5. Here we present the structure of the prototypical Salmonella enterica serovar Typhimurium pathogenicity island 1 basal body, determined using single-particle cryo-electron microscopy, with the inner-membrane-ring and outer-membrane-ring oligomers defined at 4.3 Å and 3.6 Å resolution, respectively. This work presents the first, to our knowledge, high-resolution structural characterization of the major components of the basal body in the assembled state, including that of the widespread class of outer-membrane portals known as secretins.

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Acknowledgements

We thank A. Cheung for assistance with the expression trials of the PrgH130–392 GFP-fused basal body, C. Lizak for advice on GFP variants and membrane-protein purification and C. Yip and J. Rubenstein for advice on negative-stain TEM techniques. We thank UBC Bioimaging for access to TEM infrastructure. We thank K.-M. Moon and J. Rogalski at the Michael Smith Labs Proteomics Core Facility for assistance with LC–MS/MS. We thank F. Rosell at the LMB Spectroscopy and Kinetics Hub for assistance with circular dichroism. We thank S. Miller for providing the S. Typhimurium deletion strains and plasmids, as well as the InvG antibody. This work was funded by scholarships to L.W. and J.B. from the Canadian Institutes of Health Research (CIHR) and Michael Smith Foundation of Health Research, respectively, and operating grants from CIHR to N.C.J.S. and B.B.F., and the Howard Hughes International Senior Scholar program to N.C.J.S. N.C.J.S. is a Tier I Canada Research Chair in Antibiotic Discovery.

Author information

Author notes

    • L. J. Worrall
    •  & C. Hong

    These authors contributed equally to this work.

    • J. R. C. Bergeron
    •  & T. Spreter

    Present addresses: Department of Biochemistry, University of Washington, Seattle, Washington 98195, USA (J.R.C.B.); Zymeworks, Vancouver, British Columbia V6H 3V9, Canada (T.S.).

Affiliations

  1. Department of Biochemistry and Molecular Biology and the Center for Blood Research, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada

    • L. J. Worrall
    • , M. Vuckovic
    • , J. R. C. Bergeron
    • , D. D Majewski
    • , T. Spreter
    •  & N. C. J. Strynadka
  2. CryoEM Shared Resources, Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia 20147, USA

    • C. Hong
    • , R. K. Huang
    •  & Z. Yu
  3. Michael Smith Laboratories, University of British Columbia, Vancouver, British Columbia V6T 1Z4, Canada

    • W. Deng
    •  & B. B. Finlay

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Contributions

L.J.W. performed all model building, refinement, structural analysis and modelling experiments. C.H. performed single particle cryo-EM grid preparation, data collection and map generation with input from R.K.H. and Z.Y. M.V. performed all cloning, basal body and secretin sample preparations used in the structure solution with input from L.J.W. and J.R.C.B., building on work initiated by T.S. L.J.W. and J.R.C.B. carried out negative-stain TEM analysis on basal body and secretin preps for quality control. L.J.W. and M.V. designed and made basal body mutants for secretion assays carried out by W.D. W.D. also generated/validated the invG deletion mutant. D.D.M. performed experiments probing isolated pilotin InvH and InvG S domain interactions. L.J.W. and N.C.J.S. principally wrote the manuscript with input from all.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Z. Yu or N. C. J. Strynadka.

Reviewer Information Nature thanks M. Beeby, A. Blocker, J. Rubinstein and the other anonymous reviewer(s) for their contribution to the peer review of this work.

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  1. 1.

    Supplementary Information

    This file contains original SDS-PAGE gels and anti-InvG western blots used to generate Fig. 3e (a, b), Extended Data Fig. 5f (a, b), Extended Data Fig. 8g (a, b), Extended Data Fig. 10c (c, d) and Extended Data Fig. 9a (e).

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https://doi.org/10.1038/nature20576

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