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Structure of the calcium-dependent type 2 secretion pseudopilus


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.

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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.

Author information

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.

Competing interests

The authors declare no competing financial interests.

Correspondence to Nadia Izadi-Pruneyre or Olivera Francetic.

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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.