PAAR-repeat proteins sharpen and diversify the type VI secretion system spike

Abstract

The bacterial type VI secretion system (T6SS) is a large multicomponent, dynamic macromolecular machine that has an important role in the ecology of many Gram-negative bacteria. T6SS is responsible for translocation of a wide range of toxic effector molecules, allowing predatory cells to kill both prokaryotic as well as eukaryotic prey cells1,2,3,4,5. The T6SS organelle is functionally analogous to contractile tails of bacteriophages and is thought to attack cells by initially penetrating them with a trimeric protein complex called the VgrG spike6,7. Neither the exact protein composition of the T6SS organelle nor the mechanisms of effector selection and delivery are known. Here we report that proteins from the PAAR (proline-alanine-alanine-arginine) repeat superfamily form a sharp conical extension on the VgrG spike, which is further involved in attaching effector domains to the spike. The crystal structures of two PAAR-repeat proteins bound to VgrG-like partners show that these proteins sharpen the tip of the T6SS spike complex. We demonstrate that PAAR proteins are essential for T6SS-mediated secretion and target cell killing by Vibrio cholerae and Acinetobacter baylyi. Our results indicate a new model of the T6SS organelle in which the VgrG–PAAR spike complex is decorated with multiple effectors that are delivered simultaneously into target cells in a single contraction-driven translocation event.

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Figure 1: Crystal structure of the VCA0105 PAAR-repeat protein bound to its VgrG-like partner.
Figure 2: PAAR proteins are required for full functionality of the T6SS in Vibrio cholerae and Acinetobacter baylyi.
Figure 3: The VSV-G epitope-tagged PAAR protein ACIAD2681 is secreted by A. baylyi ADP1.
Figure 4: Multiple effector translocation VgrG (MERV) model for the organization of the T6SS central spike/baseplate.

Accession codes

Primary accessions

Protein Data Bank

Data deposits

The atomic coordinates and the structure factors of the refined atomic models of gp5(VCA0018)–(VCA0105) and gp5(c1883)–(c1882) complexes were deposited to the Protein Data Bank (http://www.rcsb.org) under the accession numbers 4JIV and 4JIW, respectively.

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Acknowledgements

We thank the entire staff of the Swiss Light Source PX beamlines and V. Olieric in particular for support related to crystallographic data collection. The project was made possible by the Swiss National Science Foundation (grant 31003A_127092) and EPFL funding to P.G.L., and grants AI-026289 and AI-01845 from the NIAID to J.J.M.

Author information

M.M.S. performed the initial bioinformatic analysis that led to the identification of PAAR proteins; M.M.S. cloned all VgrG and PAAR proteins for biochemical characterization and crystallization; M.M.S. and P.G.L. designed VgrG–PAAR binding experiments; M.M.S. and S.A.B. performed gp5(VgrG)–PAAR binding experiments and purification of gp5(VgrG)–PAAR complexes; S.A.B. crystallized gp5(VgrG)–PAAR complexes and determined their crystal structures; J.J.M., M.B. and B.T.H. designed T6SS secretion and T6SS-mediated killing assay experiments involving PAAR mutants, B.T.H. and M.B. performed these experiments; B.T.H. performed the bioinformatics analysis of PAAR protein domain extensions. All authors participated in writing the manuscript.

Correspondence to John J. Mekalanos or Petr G. Leiman.

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Extended data figures and tables

Extended Data Figure 1 Design of gp5–VgrG chimaeras and analysis of their interaction with selected PAAR proteins.

a, Identification of the blunt ends of selected VgrG β-helices. The last β-strand of the known T4 gp5 structure is shown in blue. Putative β-strands terminating VgrG β-helices are in plum colour. The glycine/serine-rich motif is bold highlighted. The residue number for the first amino acid of the shown fragment and that for the C-terminal amino acid of the protein are given. Abbreviations used EC, E. coli CTF073; VC, V. cholerae O1 biovar El Tor str. N16961. b, Binding of several PAAR proteins to gp5-based VgrG-like β-helices. Two complexes for which the crystal structures are reported in this paper are highlighted with green background. Entries showing gp5 modifications that did not result in PAAR binding have a grey background. c1882* contains three mutations T28K, T64K and T90K that were made to mimic T4 gp5.4. Note that VCA0105 and VCA0284 contain threonines and serines in these positions and they bind wild-type gp5.

Extended Data Figure 2 Surface features of gp5(VgrG)–PAAR complexes and VgrG–PAAR interface.

a, b, Molecular surfaces are coloured according to their hydrophobicity with sky blue, white, and orange corresponding to the most hydrophilic, neutral and hydrophobic patches, respectively. Residue hydrophobicity values are according to the Kyte-Doolittle scale33, which is given as a coloured bar labelled ‘KD hydropathy’. Three orientations of the gp5(VgrG)–PAAR complex and an ‘open book’ view of the VgrG–PAAR interface are shown for both PAAR proteins.

Extended Data Figure 3 Main chain hydrogen-bonding network of VgrG–PAAR interface.

The dashed line rectangle in the left panel indicates the area shown enlarged in the panels on the right. The three right panels show the main chain hydrogen bonds between VgrG and PAAR proteins for three different sides of the gp5(VgrG)–PAAR complex. Residues with side chains pointing inwards and forming the VgrG–PAAR hydrophobic interface are in bold italic. Side chains are not shown for clarity. The carbon atoms of the PAAR protein are colored orange.

Extended Data Figure 4 PAAR proteins contain a Zn atom.

a, X-ray fluorescence spectra of gp5(VC0018)–VCA0105 and gp5(c1883)–c1882 crystals and their cryoprotectant solutions. The excitation wavelength is 1.0 Å (12.4 keV). The energies and peak heights for the atomic Zn and lines are taken from ref. 34. b, c, Anomalous difference Fourier maps (magenta mesh) of both crystal structures contain only one non-noise peak corresponding to the Zn atom. The VCA0105 and c1882 maps are contoured at 15.0 and 6.0 standard deviations above the mean, respectively. The corresponding noise level of the two maps is 5.1 and 4.5 standard deviations above the mean, respectively.

Extended Data Figure 5 Conserved features of PAAR proteins.

a, WebLogo35 sequence alignment of VCA0105 homologues identified with BLAST36. GenBank accession numbers of protein sequences that were used to prepare this WebLogo diagram in order of their appearance in the BLAST alignment file: 15600876, 227811731, 153212840, 417818856, 262403304, 258622274, 261212686, 260769543, 336125205, 417954065, 153801242, 424808850, 399908303, 15601050, 153817580, 254284956, 444380739, 307545498, 237731059, 197336227, 387815895, 59712519, 149377963, 120556511, 359395272, 338997855, 209695798, 338998698, 126665853, 440287404, 327412950, 145298557, 385332665, 87118978, 433087003, 433201317, 433072538, 432504178, 222155056, 262169027, 26247740, 145300690, 422833042, 432730965, 91210698, 432592619, 222156204, 432758040, 218704924, 26247745, 222156197, 417084314, 50121038, 444375671, 253990116, 423141622, 416895592, 423207453, 417628384, 331657463, 406676759, 251790158, 323491128, 421082032, 437829594, 120555573, 433076042, 145301291, 269102718, 261823483, 343512222, 343512225, 343515540, 343510421, 416895589, 145299403, 117619593, 385873501, 425070534, 343512226, 343510889, 422021929, 229523366, 262402814, 385870158, 411009627, 385869971, 258627229, 295418810, 37525853, 261819862, 307133118, 432995158, 422803770, 253987746, 253688569, 114319223, 268590359 and 261822686. The conserved PAAR motif and residues forming the Zn binding site are labelled. b, c, Pseudotrimeric organization of the three interacting PAAR motifs in VCA0105 and c1882, respectively. The buried hydrogen bonds stabilizing the fold are shown with dashed lines.

Extended Data Figure 6 Superposition of VCA0105 and c1882 PAAR structures.

Residue numbers are given at strategic positions.

Extended Data Figure 7 Quantification of Hcp secretion.

Hcp secretion was evaluated by quantifying the intensity of the Hcp band in SDS–PAGE using the Fiji software31 and normalizing it to the wild type. The presented data are the average of three experiments. The error bars correspond to one standard deviation. The mutants are labelled as in Fig. 2.

Extended Data Figure 8 Complementation analysis of heterologous PAAR proteins.

a, SDS–PAGE assay for Hcp secretion of the V. cholerae 2740-80 2 × P mutant described in Fig. 2 legend carrying a plasmid expressing endogenous or heterologous PAAR proteins. Plasmids pV105, pA0052, and pA2681 express PAAR genes vca0105 (Vibrio), aciad0052 (Acinetobacter) and aciad2681 (Acinetobacter), respectively, under the control of arabinose-inducible pBAD promoter30. N.P. stands for no plasmid. Cells were grown in the presence of 0.001%, 0.01% and 0.1% arabinose for 4 h. Both panels show a typical representative of three identical experiments. b, E. coli MG1655 killing by the V. cholerae 2740-80 2 × P mutant that carries a plasmid expressing endogenous and heterologous PAAR proteins. The complementation efficiency is calculated as the log10 ratio of E. coli recovered after competition with the wild type strain of V. cholerae 2740-80 and its 2 × P mutant supplemented with the indicated plasmid. The data represent the average of three experiments; error bars correspond to one standard deviation.

Extended Data Figure 9 Bioinformatic analysis of PAAR proteins.

a, Domain organization of all known bacterial (non-phage) PAAR proteins. b, Relative abundance of the seven distinct domain organizations. c, Predicted functions of the C-terminal domains. TTR stands for transthyretin domain.

Extended Data Table 1 Crystallographic data collection and refinement statistics.

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This file contains Supplementary Notes, which relate to Extended Data Figure 5. (PDF 115 kb)

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Shneider, M., Buth, S., Ho, B. et al. PAAR-repeat proteins sharpen and diversify the type VI secretion system spike. Nature 500, 350–353 (2013) doi:10.1038/nature12453

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