Structure of a bacterial Rhs effector exported by the type VI secretion system

Abstract

The type VI secretion system (T6SS) is a widespread protein export apparatus found in Gram-negative bacteria. The majority of T6SSs deliver toxic effector proteins into competitor bacteria. Yet, the structure, function, and activation of many of these effectors remains poorly understood. Here, we present the structures of the T6SS effector RhsA from Pseudomonas protegens and its cognate T6SS spike protein, VgrG1, at 3.3 Å resolution. The structures reveal that the rearrangement hotspot (Rhs) repeats of RhsA assemble into a closed anticlockwise β-barrel spiral similar to that found in bacterial insecticidal Tc toxins and in metazoan teneurin proteins. We find that the C-terminal toxin domain of RhsA is autoproteolytically cleaved but remains inside the Rhs ′cocoon′ where, with the exception of three ordered structural elements, most of the toxin is disordered. The N-terminal ′plug′ domain is unique to T6SS Rhs proteins and resembles a champagne cork that seals the Rhs cocoon at one end while also mediating interactions with VgrG1. Interestingly, this domain is also autoproteolytically cleaved inside the cocoon but remains associated with it. We propose that mechanical force is required to remove the cleaved part of the plug, resulting in the release of the toxin domain as it is delivered into a susceptible bacterial cell by the T6SS.

Mechanism of threonine ADP-ribosylation of F-actin by a Tc toxin

“…This also explains why TcHVR was neither resolved in cryo-EM structures nor X-ray structures of TcB-TcC cocoons from P. luminescens and Y. entomophaga . Interestingly, the effector could also not be resolved in Rhs toxins from Pseudomonas protegens and Photorhabdus laumondii 39,40 . Similarly to TccC3, the N-terminal part of Rhs forms a cocoon that encapsulates the C-terminal effector region.…”

Archaeal type six secretion system mediates contact-dependent antagonism

“…From tomography data and subtomogram averaging, we did not observe extra densities within the lumen of the inner tube. We therefore postulate that these toxins attach to the T6SS spike, as was previously shown (41,42).…”

TomoTwin: Generalized 3D Localization of Macromolecules in Cryo-electron Tomograms with Structural Data Mining

“…As TomoTwin is trained entirely on simulated data, it is paramount to investigate its ability to pick proteins of interest in experimental tomograms. To evaluate this, cryo-ET was performed on a sample containing a mixture of three proteins, namely apoferritin 34 , the Type VI secretion effector RhsA from Pseudomonas protegens 34 , and the Tc toxin A component TcdA1 from Photorhabdus luminescens 35 as well as liposomes (DOPC/POPC) (Fig. 3a).…”