Mounting, structure and autocleavage of a type VI secretion-associated Rhs polymorphic toxin

Abstract

Bacteria have evolved toxins to outcompete other bacteria or to hijack host cell pathways. One broad family of bacterial polymorphic toxins gathers multidomain proteins with a modular organization, comprising a C-terminal toxin domain fused to a N-terminal domain that adapts to the delivery apparatus. Polymorphic toxins include bacteriocins, contact-dependent growth inhibition systems, and specialized Hcp, VgrG, PAAR or Rhs Type VI secretion (T6SS) components. We recently described and characterized Tre23, a toxin domain fused to a T6SS-associated Rhs protein in Photorhabdus laumondii, Rhs1. Here, we show that Rhs1 forms a complex with the T6SS spike protein VgrG and the EagR chaperone. Using truncation derivatives and cross-linking mass spectrometry, we demonstrate that VgrG-EagR-Rhs1 complex formation requires the VgrG C-terminal β-helix and the Rhs1 N-terminal region. We then report the cryo-electron-microscopy structure of the Rhs1-EagR complex, demonstrating that the Rhs1 central region forms a β-barrel cage-like structure that encapsulates the C-terminal toxin domain, and provide evidence for processing of the Rhs1 protein through aspartyl autoproteolysis. We propose a model for Rhs1 loading on the T6SS, transport and delivery into the target cell.

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.…”

Two Type VI Secretion DNase Effectors are Utilized for Interbacterial Competition in the Fish Pathogen Pseudomonas plecoglossicida

“…In addition, VgrG, EagR, and Rhs effector counterparts encoded between the two gene clusters are also highly homologous ( Supplementary Figures S1–S3 ), indicating that the two vgrG clusters are likely the result of a gene duplication event or are acquired from close relative by gene transfer. Together, these findings suggest that the proteins encoded in the vgrG-2 and vgrG-5 clusters may form VgrG-EagR-Rhs complexes of sufficient structural similarity to interact with the tube and baseplate of the same T6SS system (namely, T6SS-2) for toxin delivery ( Jurėnas et al, 2021 ).…”

“…Some of the T6SS adaptor proteins are those belonging to the DUF1795, DUF2169, and DUF4123 families. DUF1795 family proteins, termed Eag (effector-associated gene) adaptors, bind to the N-terminal half of PAAR-containing T6SS effectors, acting as a chaperone to stabilize the transmembrane domains of “specialized” effectors during the translocation across bacterial cells ( Diniz et al, 2015 ; Whitney et al, 2015 ; Jurėnas et al, 2021 ). DUF2169 and DUF4123 family proteins seem to be less prevalent and were shown to assist the loading of “cargo” effectors onto the VgrG spike ( Unterweger et al, 2015 ; Bondage et al, 2016 ; Pei et al, 2020 ).…”

Et tu, Neisseria? Conflicts of Interest Between Neisseria Species

“…The RHS domain between the N-and C-terminus contains several repeating motifs, which when folded form a bsheet cage around the toxic domain (Günther et al, 2022). At the border between these three domains are dual autoproteolytic DPxGL motifs (Jureṅas et al, 2021). It is not clear when this cleavage occurs, but since the RHS domain forms a protective shell around the C-terminal toxin, and since the effector needs to be attached to the needle, which happens via the N-terminus, proteolysis is thought to occur after secretion (Jureṅas et al, 2021).…”

“…At the border between these three domains are dual autoproteolytic DPxGL motifs (Jureṅas et al, 2021). It is not clear when this cleavage occurs, but since the RHS domain forms a protective shell around the C-terminal toxin, and since the effector needs to be attached to the needle, which happens via the N-terminus, proteolysis is thought to occur after secretion (Jureṅas et al, 2021). Secretion of the toxin-loaded needle happens first by assembly of the basal plate spanning the inner membrane.…”