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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 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.

SUBMITTER: Shneider MM

PROVIDER: S-EPMC3792578 | biostudies-literature | 2013 Aug

REPOSITORIES: biostudies-literature

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

Shneider Mikhail M MM Buth Sergey A SA Ho Brian T BT Basler Marek M Mekalanos John J JJ Leiman Petr G PG

Nature 20130807 7462

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 tri ...[more]

PMID: 23925114

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Project description:The Type VI secretion system (T6SS) is widespread among bacterial pathogens and acts as an effective weapon against competitor bacteria and eukaryotic hosts by delivering toxic effector proteins directly into target cells. The T6SS utilises a bacteriophage-like contractile machinery to expel a puncturing device based on a tube of Hcp topped with a VgrG spike, which can be extended by a final tip from a PAAR domain-containing protein. Effector proteins are believed to be delivered by specifically associating with particular Hcp, VgrG or PAAR proteins, either covalently ('specialised') or non-covalently ('cargo' effectors). Here we used the T6SS of the opportunistic pathogen Serratia marcescens, together with integratecd genetic, proteomic and biochemical approaches, to elucidate the role of specific VgrG and PAAR homologues in T6SS function and effector specificity, revealing new aspects and unexpected subtleties in effector delivery by the T6SS. We identified effectors, both cargo and specialised, absolutely dependent on a particular VgrG for delivery to target cells, and discovered that other cargo effectors can show a preference for a particular VgrG. The presence of at least one PAAR protein was found to be essential for T6SS function, consistent with designation as a 'core' T6SS component. We showed that specific VgrG-PAAR combinations are required to assemble a functional T6SS and that the three distinct VgrG-PAAR assemblies in S. marcescens exhibit distinct effector specificity and efficiency. Unexpectedly, we discovered that two different PAAR-containing Rhs proteins can functionally pair with the same VgrG protein. Showing that accessory EagR proteins are involved in these interactions, native VgrG-Rhs-EagR complexes were isolated and specific interactions between EagR and cognate Rhs proteins identified. This study defines an essential yet flexible role for PAAR proteins in the T6SS and highlights the existence of distinct versions of the machinery with differential effector specificity and efficiency of target cell delivery.

Project description:Bacteria are constantly competing to colonize crowded ecological niches, such as the human gut. The type VI secretion system (T6SS) is a critical bacterial weapon in this warfare. It resembles a crossbow with a poisoned arrow allowing bacteria to inject toxic effectors directly into target cells. This machinery is formed by an envelope-spanning complex which recruits the baseplate, an assembly platform allowing the polymerization of a contractile structure. The tail consists of a tube surrounded by a sheath and topped by the needle complex composed of the VgrG and PAAR proteins. In the enteric pathogen enteroaggregative Escherichia coli (EAEC), the Tle1 phospholipase toxin ensures the antibacterial activity of the T6SS-1. For its transport, Tle1 interacts directly with the trimeric spike protein VgrG. However, the importance and the function of the tip protein PAAR in the T6SS remain unclear. Here, we characterized the PAAR protein of EAEC using biochemical, fluorescence microscopy, and antibacterial competition approaches. Using pull-down assays and cryo-electron microscopy analysis of the (VgrG)3-(Tle1)3-PAAR complex, we show that PAAR tops and closes the β-prism structure of the VgrG spike. The PAAR protein structure is further tightened by the zinc atom coordinated via conserved residues essential for its function. We provide evidence that PAAR is necessary for T6SS-1-mediated killing due to its requirement for proper T6SS baseplate assembly and further sheath polymerization. Our results suggest that the PAAR protein is an essential component of T6SS. IMPORTANCE The type VI secretion system (T6SS) is a bacterial contractile injection system involved in bacterial competition by the delivery of antibacterial toxins. The T6SS consists of an envelope-spanning complex that recruits the baseplate, allowing the polymerization of a contractile tail structure. The tail is a tube wrapped by a sheath and topped by the tip of the system, the VgrG spike/PAAR complex. Effectors loaded onto the puncturing tip or into the tube are propelled in the target cells upon sheath contraction. The PAAR protein tips and sharpens the VgrG spike. However, the importance and the function of this protein remain unclear. Here, we provide evidence for association of PAAR at the tip of the VgrG spike. We also found that the PAAR protein is a T6SS critical component required for baseplate and sheath assembly.

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

Publications

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

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