PAAR-repeat proteins sharpen and diversify the type VI secretion system spike - PubMed (original) (raw)

. 2013 Aug 15;500(7462):350-353.

doi: 10.1038/nature12453. Epub 2013 Aug 7.

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

Mikhail M Shneider et al. Nature. 2013.

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 cells. 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 spike. 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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Figures

Figure 1

Figure 1

Crystal structure of the VCA0105 PAAR-repeat protein bound to its VgrG-like partner. a, Schematic representation of the conserved domains comprising the VgrG-PAAR complex. The last strands of the β-helix that form the PAAR binding site are in light blue. Gray arrow shows the fragment roughly corresponding to the crystal structure. b, Molecular surface representation of the gp5_VCA0018-VCA0105 complex crystal structure. Each protein chain is labeled with its own color. c, Ribbon diagram of the gp5_VCA0018- VCA0105 complex. d, The polypeptide chain of the VCA0105 PAAR protein is colored in rainbow colors with N terminus in blue and C terminus in red. Residues responsible for Zn binding are labeled.

Figure 2

Figure 2

PAAR proteins are required for full functionality of the T6SS in Vibrio cholerae and Acinetobacter baylyi. a, Recovery of viable E. coli MG1655 after co-incubation with A. baylyi ADP1 (WT) and its T6SS and PAAR genes knockout mutants. The following genes were inactivated in the mutants shown: T6S¯ – aciad2688 to aciad2694; 2681¯ – aciad2681; 0051-52¯ – both aciad0051 and aciad0052; 3×P¯ – all three PAAR genes aciad0051, aciad0052 and aciad2681. Right subpanel: Leaky, basal expression of the aciad2681 PAAR gene from plasmid pMMB67EH, labeled as p2681, restores the killing defect in the triple PAAR mutant. b, Recovery of E. coli MG1655 colony forming units after co-incubation with V. cholerae 2740-80 and its T6SS and PAAR genes knockout mutants, which are labeled as follows: T6S¯ – vipA; 105¯ – vca0105; 284¯ – vca0284; 2×P¯ – vca0105 and vca0284. Symbols *, **, and *** indicate deviations from the WT with p-values of 6×10−3, 8×10−3, and 5×10−7, respectively, for a sample size of 8. Error bars represent one standard deviation. c, SDS-PAGE assay for T6SS-dependent secretion of Hcp proteins by the parental strains and T6SS and PAAR genes knockout mutants. Panels a and c show one out of three experiments with similar outcomes.

Figure 3

Figure 3

The vsvG epitope-tagged PAAR protein ACIAD2681 is secreted by A. baylyi ADP1. Left panels: T6SS-dependent secretion of vsvG epitope-tagged ACIAD2681 expressed from plasmid pMMB67EH. Right panel: vsvG epitope-tagged ACIAD2681 fully restores the Hcp secretion defect of the triple knockout PAAR mutant of A. baylyi ADP1. The mutants are labeled as in Fig. 2. A representative of three identical experiments was chosen for each panel.

Figure 4

Figure 4

Multiple Effector TRanslocation VgrG (MERV) model for the organization of the T6SS central spike/baseplate. Effectors are predicted to be loaded onto the spike complex by five distinct mechanisms: 1) C-terminal extensions of the VgrG spike; 2) Non-covalent binding to the VgrG spike; 3) N- or C-terminal extensions of the PAAR protein; 4) Non- covalent binding to the PAAR protein or its extension domains; 5) Incorporation into the cavity formed by the gp27 domain of VgrG. A single T6SS sheath contraction event translocates the VgrG spike with all of its cargo proteins into a nearby target cell. Other proteins making up the T6SS “baseplate” are not labeled but presumably reside within or attached to the inner and outer membranes and peptidoglycan layer (IM, OM, and PG, respectively).

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