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Structure of the calcium-dependent type 2 secretion pseudopilus

Nature Microbiology volume 2, pages 1686–1695 (2017)Cite this article

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Abstract

Many Gram-negative bacteria use type 2 secretion systems (T2SSs) to secrete proteins involved in virulence and adaptation. Transport of folded proteins via T2SS nanomachines requires the assembly of inner membrane-anchored fibres called pseudopili. Although efficient pseudopilus assembly is essential for protein secretion, structure-based functional analyses are required to unravel the mechanistic link between these processes. Here, we report an atomic model for a T2SS pseudopilus from Klebsiella oxytoca, obtained by fitting the NMR structure of its calcium-bound subunit PulG into the ~5-Å-resolution cryo-electron microscopy reconstruction of assembled fibres. This structure reveals the comprehensive network of inter-subunit contacts and unexpected features, including a disordered central region of the PulG helical stem, and highly flexible C-terminal residues on the fibre surface. NMR, mutagenesis and functional analyses highlight the key role of calcium in PulG folding and stability. Fibre disassembly in the absence of calcium provides a basis for pseudopilus length control, essential for protein secretion, and supports the Archimedes screw model for the type 2 secretion mechanism.

Microorganisms profoundly transform their environment by colonizing biotic and abiotic surfaces and assimilating nutrients from organic and mineral sources. In many prokaryotes, nanomachines of the type 4 filament superfamily1 mediate adaptive functions by promoting cell adhesion, motility and macromolecular transport. The type 4 filament assembly systems, including archaeal flagella2, bacterial type 4 pili (T4P) and T2SSs3, use a conserved mechanism to polymerize fibres from protein subunits initially localized in the plasma membrane. In Gram-negative bacteria, T2SSs promote assembly of periplasmic fibres called pseudopili to drive secretion of specific folded proteins, including macromolecule hydrolases, toxins, adhesins or cytochromes with key roles in virulence and niche survival4.

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Fig. 1: Calcium is required for PulG stability, pseudopilus assembly and stability.
Fig. 2: The effect of calcium on PulGp folding and identification of calcium-coordinating residues.
Fig. 3: NMR structure of PulGp in the calcium-bound state.
Fig. 4: Morphology of the Klebsiella T2SS pseudopilus PulGCC filament by cryoEM and reconstruction at ~5 Å resolution.
Fig. 5: Structure of the PulGCC pilus.

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Acknowledgements

This work was funded by the Institut Pasteur, the Centre National de la Recherche Scientifique (CNRS), the French Agence Nationale de la Recherche (ANR-14-CE09-0004), the European Union FP7-IDEAS-ERC 294809 (to M. Nilges) and the NIH R35GM122510 (to E.H.E.). We thank L. Khoury for help in sample preparation, and the Plateforme de Biophysique Moléculaire of Institut Pasteur and B. Baron for assistance in circular dichroism experiments. We are grateful to M. Delepierre and D. Ladant for support and interest in this work. We acknowledge N. Morellet and financial support from the TGIR-RMN-THC Fr3050 CNRS.

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  1. Aracelys López-Castilla, Jenny-Lee Thomassin and Benjamin Bardiaux contributed equally to this work.

Authors and Affiliations

  1. NMR of Biomolecules Unit, Department of Structural Biology and Chemistry, Institut Pasteur, CNRS UMR3528, 28 rue du Dr Roux, 75724, Paris, France

    Aracelys López-Castilla & Nadia Izadi-Pruneyre

  2. Biochemistry of Macromolecular Interactions Unit, Department of Structural Biology and Chemistry, Institut Pasteur, CNRS ERL6002, 28 rue du Dr Roux, 75724, Paris, France

    Jenny-Lee Thomassin, Mangayarkarasi Nivaskumar & Olivera Francetic

  3. Structural Bioinformatics Unit, Department of Structural Biology and Chemistry, CNRS UMR3528, Institut Pasteur, 25 rue du Dr Roux, 75724, Paris, France

    Benjamin Bardiaux & Michael Nilges

  4. Department of Biochemistry and Molecular Genetics, University of Virginia, Charlottesville, VA 22908, USA

    Weili Zheng, Xiong Yu & Edward H. Egelman

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  1. Aracelys López-Castilla
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Contributions

Conceived and designed the experiments: A.L.-C., J.-L.T., B.B., M. Nil., E.H.E., N.I.-P. and O.F. Performed the experiments: A.L.-C., J.-L.T., B.B., W.Z., X.Y., M. Niv. Analysed the data and wrote the manuscript: A.L.-C, J.-L.T., B.B., W.Z., M. Nil., E.H.E., N.I.-P. and O.F.

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Correspondence to Nadia Izadi-Pruneyre or Olivera Francetic.

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López-Castilla, A., Thomassin, JL., Bardiaux, B. et al. Structure of the calcium-dependent type 2 secretion pseudopilus. Nat Microbiol 2, 1686–1695 (2017). https://doi.org/10.1038/s41564-017-0041-2

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