Protein refolding is required for assembly of the type three secretion needle

Journal name:
Nature Structural & Molecular Biology
Volume:
17,
Pages:
788–792
Year published:
DOI:
doi:10.1038/nsmb.1822
Received
Accepted
Published online

Abstract

Pathogenic Gram-negative bacteria use a type three secretion system (TTSS) to deliver virulence factors into host cells. Although the order in which proteins incorporate into the growing TTSS is well described, the underlying assembly mechanisms are still unclear. Here we show that the TTSS needle protomer refolds spontaneously to extend the needle from the distal end. We developed a functional mutant of the needle protomer from Shigella flexneri and Salmonella typhimurium to study its assembly in vitro. We show that the protomer partially refolds from α-helix into β-strand conformation to form the TTSS needle. Reconstitution experiments show that needle growth does not require ATP. Thus, like the structurally related flagellar systems, the needle elongates by subunit polymerization at the distal end but requires protomer refolding. Our studies provide a starting point to understand the molecular assembly mechanisms and the structure of the TTSS at atomic level.

At a glance

Figures

  1. Polymerization of a functional TTSS protomer.
    Figure 1: Polymerization of a functional TTSS protomer.

    (a) SDS-PAGE of PrgI, PrgI*, MxiH and MxiH* expressed in E. coli. Double mutants were detected in soluble (SN) and insoluble (P) fractions, whereas wild-type protomer was almost insoluble (arrow). (b) Epithelial cell invasion assay showed wild type activity of S. typhimurium protomer knockouts complemented with either wild-type gene (pprgI) or protomer mutant (pprgI*). Percent bacterial invasion is logarithmically scaled. Deletion (pprgI ΔC5) or fusion (pprgI ΔC-His) of residues at the PrgI C terminus, as well as deletion of the N-terminal eight residues (pprgI ΔN8), abolished cell invasion. (c) TEM image of negatively stained needles formed in vitro by purified PrgI* (scale bar, 200 nm). (d) Time-dependent monomer conversion of PrgI* (solid line) versus needle growth (dashed line) monitored by DLS. Arrow, end of the lag phase.

  2. In vitro assembly of TTSS needles.
    Figure 2: In vitro assembly of TTSS needles.

    (a) Purified PrgI* was incubated with isolated TTSS (arrow) from S. typhimurium and analyzed by TEM (scale bar, 0.1 μm). (b) TTSS from an S. flexneri strain were incubated with purified MxiH* and visualized (scale bar, 1 μm). Lower images show enlarged regions (scale bar, 0.1 μm). (c) Length of needles of isolated TTSS from S. flexneri without addition of purified protomer and of elongated needles obtained after incubation of TTSS from S. flexneri with purified MxiH* or purified IpaD and MxiH*. Error bars, s.d. (n = 20).

  3. Protomer refolding during assembly of the TTSS needle.
    Figure 3: Protomer refolding during assembly of the TTSS needle.

    (a) X-ray crystal and NMR solution structure of PrgI*. Mutated residues (V65A, V67A) and isoleucines are highlighted in the X-ray crystal structure. Five C-terminal residues essential for PrgI* function are marked in green. Functionally dispensable residues at the N terminus are highlighted in red in the NMR structure. (b) Time course of FTIR difference spectra collected during polymerization for a period of 28 h. Spectral changes indicate α-helix–to–β-strand conversion of protomer molecules over time. Line color changes with increasing time from red to purple. (c) Solid-state NMR (13C,13C) correlation spectrum of PrgI* in needles (black contours) in comparison with chemical-shift correlations corresponding to solution-state NMR assignments (red crosses). Correlations labeled with blue residue codes are unique to solid-state spectra and correspond to β-strand backbone structure.

  4. Assembly of the TTSS needle.
    Figure 4: Assembly of the TTSS needle.

    (a) Proposed secondary structure of PrgI* before (stage I) polymerization and after (stage II) forming needles (α-helix, bar; β-strand, arrow; random coil, line). Hatched regions in stage I correspond to secondary structure with higher flexibility. For dashed regions in stage II, no unambiguous resonance assignments are available. (b) Model of needle constitution. Cytosolic protomer is transported by the TTSS to the tip of the growing needle.

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Protein Data Bank

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Author information

  1. These authors contributed equally to this work.

    • Ömer Poyraz,
    • Holger Schmidt &
    • Karsten Seidel

Affiliations

  1. Max-Planck-Institute for Infection Biology, Cellular Microbiology, Berlin, Germany.

    • Ömer Poyraz,
    • Hezi Tenenboim,
    • Arturo Zychlinsky &
    • Michael Kolbe
  2. Max-Planck-Institute for Biophysical Chemistry, NMR based Structural Biology, Göttingen, Germany.

    • Holger Schmidt,
    • Karsten Seidel,
    • Christian Ader,
    • Marc Baldus,
    • Adam Lange &
    • Christian Griesinger
  3. BAM Federal Institute for Material Research and Testing, Berlin, Germany.

    • Friedmar Delissen &
    • Andreas F Thünemann
  4. Max-Planck-Institute for Infection Biology, Core Facility Microscopy, Berlin, Germany.

    • Christian Goosmann &
    • Britta Laube
  5. Present addresses: BASF SE, Ludwigshafen, Germany (K.S.), Bijvoet Center for Biomolecular Research, Utrecht University, Utrecht, The Netherlands (C.A. and M.B.) and Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden (Ö.P.).

    • Ömer Poyraz,
    • Karsten Seidel,
    • Christian Ader &
    • Marc Baldus

Contributions

Ö.P. cloned constructs and purified protomer for every experiment, crystallized, collected, processed and refined X-ray diffraction data and performed cellular assays; H.S. collected, processed and analyzed liquid-state NMR data and performed FTIR experiments and ThioflavinT binding assays; F.D. performed and analyzed DLS and X-ray fiber diffraction experiments; K.S. and C.A. collected, processed and analyzed solid-state NMR data; H.T. and Ö.P. purified and performed in vitro growth experiments with TTSS; C. Goosmann performed TEM studies; A.L. and M.B. designed and analyzed solid-state NMR experiments; C. Griesinger designed and analyzed liquid-state NMR experiments; A.T. designed and analyzed DLS and X-ray fiber diffraction experiments; V.B. designed TEM experiments; A.Z. designed functional and structural experiments; M.K. conceived this study, designed functional and TEM experiments, collected, refined and analyzed X-ray diffraction data and wrote the paper; all authors discussed the results and commented on the manuscript.

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The authors declare no competing financial interests.

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