Three-dimensional electron microscopy reconstruction and cysteine-mediated crosslinking provide a model of the T3SS needle tip complex

Martin Cheung, Dakang Shen, Fumiaki Makino, Takayuki Kato, A Dorothea Roehrich, Isabel Martinez-Argudo, Matthew L Walker, Isabel Murillo, Xia Liu, Maria Pain, James Brown, Gordon Frazer, Judith Mantell, Petros Mina, Thomas Todd, Richard B Sessions, Keiichi Namba, Ariel J Blocker

Research output: Contribution to journalArticle (Academic Journal)

24 Citations (Scopus)

Abstract

Type III secretion systems are found in many Gram-negative bacteria. They are activated by contact with eukaryotic cells and inject virulence proteins inside them. Host cell detection requires a protein complex located at the tip of the device's external injection needle. The tip complex (TC) is composed of IpaD, a hydrophilic protein, and IpaB, a hydrophobic protein, which later forms part of the injection pore in the host membrane. Here we used labelling and crosslinking methods to show that TCs from a ΔipaB strain contain 5 IpaD subunits while the TCs from wild-type can also contain 1 IpaB and 4 IpaD subunits. Electron microscopy followed by single particle and helical image analysis was used to reconstruct three-dimensional images of TCs at ∼20 Å resolution. Docking of an IpaD crystal structure, constrained by the crosslinks observed, reveals TC organisation is different from that of all previously proposed models. Our findings suggest new mechanisms for tip complex assembly and function. The tip complex is the only site within these secretion systems targeted by disease-protecting antibodies. By suggesting how these act, our work will allow improvement of prophylactic and therapeutic strategies.

Original languageEnglish
Pages (from-to)31-50
JournalMolecular Microbiology
Volume95
DOIs
Publication statusPublished - 29 Oct 2014

Bibliographical note

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  • Projects

    Bacterial type III secretion systems: from structure to function

    Blocker, A. J.

    28/01/1228/05/15

    Project: Research

    GA0401595

    Blocker, A. J.

    1/02/081/02/09

    Project: Research

    Datasets

    Electron microscopy 3DR of T3SS needle tip complex

    Kato, T. (Creator), Blocker, A. (Creator), Makino, F. (Creator), Cheung, M. (Creator) & Fernando, V. (Creator), University of Bristol, 29 Oct 2014

    Dataset

    Equipment

  • Cite this

    Cheung, M., Shen, D., Makino, F., Kato, T., Roehrich, A. D., Martinez-Argudo, I., Walker, M. L., Murillo, I., Liu, X., Pain, M., Brown, J., Frazer, G., Mantell, J., Mina, P., Todd, T., Sessions, R. B., Namba, K., & Blocker, A. J. (2014). Three-dimensional electron microscopy reconstruction and cysteine-mediated crosslinking provide a model of the T3SS needle tip complex. Molecular Microbiology, 95, 31-50. https://doi.org/10.1111/mmi.12843