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.…”

A novel variant of theListeria monocytogenestype VII secretion system EssC component is associated with an Rhs toxin

“…Many Rhs-repeat proteins are anti-bacterial toxins that can be secreted via a number of routes, including the Gram-negative Type VI secretion system and by the Sec pathway in Gram-positive bacteria, dependent upon the targeting information that is present (50, 51). The Rhs repeats form a filamentous ‘cocoon’ that encases a C-terminal toxin domain (52).…”

“…The maximum size of the T7SS secretion pore is unclear as cryo EM structures of the mycobacterial T7SSa are of the closed complexes (7, 9), but modelling studies based on the related FtsK hexamer estimates a channel of approximately 30 Å (10). LrhA is the largest T7SSb substrate identified to date and is likely too large to be exported in a folded state; for example a typical Rhs cocoon such as that of Pseudomonas protegens RhsA is 86 x 65 Å (52). The Sec-dependent Rhs proteins would also be secreted unfolded and assume their 3D structure in the extracellular environment (51).…”

T6SS-mediated competitive exclusion amongPantoea agglomeransassociated with plants

“…The last two effector/immunity pairs that we identified in the 5’ region of the T6SS cluster 1 are the Pse2/Psi2 effector/immunity pair [29] in eight of the eleven strains and a putative Tre1/Tri1 effector/immunity pair present in only strain T6. The Tre1/Tri1 pair follows the canonical architecture observed in other T6SS systems with an upstream located cognate vgrG homolog (T6SS spike protein) followed by a specific Eag adaptor protein encoding gene [42,43].…”