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

Abstract

The bacterial type VI secretion system (T6SS) is a large multi-component, dynamic macromolecular machine that plays 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 cells1-5. 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 function to sharpen the tip of the VgrG spike. We demonstrate that PAAR proteins are essential for T6SS- mediated secretion and target cell killing by Vibrio cholerae and Acinetobacter baylyi. Our results suggest 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.

Cryo‐ EM reconstruction of Type VI secretion system baseplate and sheath distal end

“…This is consistent with sizes of known effectors: 129‐kDa VgrG‐1, 113‐kDa VgrG‐3, or 155‐kDa pair VasX‐VasW (Dong et al , 2013; Joshi et al , 2017). Since 77‐kDa VgrG‐2 with no extension domain is required for T6SS assembly (Appendix Fig S3; Pukatzki et al , 2007), there is also enough space for effectors interacting with VgrGs, such as 72‐kDa TseL (Dong et al , 2013), in agreement with the model that many effectors bind to the VgrG/PAAR spike (Fig EV6; Shneider et al , 2013). However, the repertoire of secreted effectors is large (Durand et al , 2014; Alcoforado Diniz et al , 2015; Hachani et al , 2016), and therefore, it is likely that the overall shape of T6SS baseplate will vary between organisms.…”

“…Overall dimensions of the baseplate are 182 Å in the narrow part, 240 Å in the broad part below the sheath–tube, and 195 Å in height including the protruding tip complex (Fig 2A and B). The central tip protrudes ~40 Å below base of the cone and clearly resembles a central spike present in all baseplates of phages with contractile tails (Browning et al , 2012; Leiman & Shneider, 2012; Schwarzer et al , 2012; Harada et al , 2013; Shneider et al , 2013; Habann et al , 2014; Taylor et al , 2016). …”

“…The T6SS spike complex is proposed to be formed by a VgrG trimer and a single PAAR protein (Leiman et al , 2009; Shneider et al , 2013). The central spike density has indeed well‐resolved features in the threefold symmetry reconstruction (Appendix Fig S1A).…”

“…T6SS effectors can be attached to the spike by several different mechanisms (Shneider et al , 2013). Mass spectrometry analysis identified T6SS effectors VasX and TseL associated with the non‐contractile sheaths (Tables EV1, EV2 and EV3).…”

Baseplate assembly of phage Mu: Defining the conserved core components of contractile-tailed phages and related bacterial systems

“…Consistent with this finding, the T6SS invariably features BH2-BS fusion proteins, and PVCs often do. Effectors for the T6SS are frequently fused to the BS or a PAAR (proline-alanine-alanine-arginine)-repeat protein bound to it (28). These distinctive properties may explain why BH2-BS fusion proteins are rare for phage tails and common for T6SSs and PVCs.…”