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

['Patrick Günther', 'Department Of Structural Biochemistry', 'Max Planck Institute Of Molecular Physiology', 'Dortmund', 'Dennis Quentin', 'Shehryar Ahmad', 'Department Of Biochemistry', 'Biomedical Sciences', 'Mcmaster University', 'Hamilton']

Date: 2022-02 

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.

Bacteria have developed a variety of strategies to compete for nutrients and limited resources. One system widely used by Gram-negative bacteria is the T6 secretion system which delivers a plethora of effectors into competing bacterial cells. Known functions of effectors are degradation of the cell wall, the depletion of essential metabolites such as NAD + or the cleavage of DNA. RhsA is an effector from the widespread plant-protecting bacteria Pseudomonas protegens. We found that RhsA forms a closed cocoon similar to that found in bacterial Tc toxins and metazoan teneurin proteins. The effector cleaves its polypeptide chain by itself in three pieces, namely the N-terminal domain including a seal, the cocoon and the actual toxic component which potentially cleaves DNA. The toxic component is encapsulated in the large cocoon, so that the effector producing bacterium is protected from the toxin. In order for the toxin to exit the cocoon, we propose that the seal, which closes the cocoon at one end, is removed by mechanical forces during injection of the effector by the T6 secretion system. We further hypothesize about different scenarios for the delivery of the toxin into the cytoplasm of the host cell. Together, our findings expand the knowledge of the mechanism of action of the T6 secretion system and its essential role in interbacterial competition.

Funding: This work was supported by the Max Planck Society (to S.R.) and a Discovery Grant from the Natural Sciences and Engineering Research Council of Canada (RGPIN-2017–05350 to J.C.W.). J.C.W. is the Canada Research Chair in Molecular Microbiology and holds an Investigators in the Pathogenesis of Infectious Disease Award from the Burroughs Wellcome Fund. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Data Availability: The cryo-EM maps of VgrG1 and RhsA have been deposited in the Electron Microscopy Data Bank (EMDB) under the accession codes of EMD-13843 and EMD-13867, respectively. The refined models for VgrG1 and RhsA were uploaded in the PDB and the entries have the IDs 7Q5P and 7Q97, respectively. The original data of the de novo protein sequencing is available inprovided as supplementary data (the S1 Dataset ).

In this work, we present high-resolution structures of the T6SS effector RhsA and its cognate VgrG1 protein at resolutions of 3.3 Å. The structure of VgrG1 is nearly identical to our previously determined VgrG1 structure from P. aeruginosa [ 16 ], indicating a high structural conservation of VgrG proteins. The structure of RhsA reveals that the Rhs-repeats of RhsA form a cocoon similar to that of the B and C components of Tc toxins and teneurins. However, unlike the cocoon of Tc toxins, which are capped by the aforementioned β-propeller domain, RhsA is closed at its N-terminus by a plug domain that connects the cocoon to its N-terminal PAAR domain. The plug domain is held in place via hydrophobic interactions with the Rhs cocoon and its eventual opening is the most likely event for the toxin domain to be released from the barrel. Our structural and biochemical findings show that the plug domain is autoproteolytically cleaved inside the cocoon. The cleaved 304 N-terminal residues, comprising the prePAAR motif, TMD, and PAAR domain also include an N-terminal seal peptide of the Rhs plug and an anchor helix. This helix interacts extensively with the inner surface of the cocoon and is likely responsible for keeping the cleaved N-terminal 304 residues physically associated with the cocoon. In addition, we find that the RhsA C-terminal toxin domain is also cleaved via a canonical Rhs aspartyl autoprotease and that this cleavage event is required for RhsA-dependent bacterial killing. Taken together, our results provide novel insights into the architecture and mechanism of action of Rhs effectors exported by the T6SS.

The Rhs repeat-containing cocoon TcBC interacts through the β-propeller domain of TcB with TcA. The propeller acts as a gate that opens upon A component binding, allowing the HVR to enter the translocation channel of TcA [ 32 ]. The penetration of the target cell membrane through the A component is triggered by either a shift to higher or lower pH [ 26 ]. Membrane penetration opens the translocation channel of TcA, which permits subsequent HVR release into the prey cell cytosol [ 26 , 33 ]. A similarly detailed toxin release mechanism has not been reported for the bacteria targeting T6SS Rhs effectors [ 3 ] and structures of T6SS-exported Rhs proteins have not been determined to date.

Tc toxins consist of three components: a 1.4 MDa homopentameric A component (TcA), a ~170 kDa TcB subunit and a ~100 kDa TcC subunit [ 26 ]. TcA acts as membrane permeation and translocation device, while TcB and TcC together form a large hollow cocoon composed of Rhs repeats. The C-terminal extension of the TcC subunit lies inside the cocoon and is autocatalytically processed by an aspartyl protease [ 26 ]. While the Rhs repeat-containing region is highly conserved among T6SS Rhs effectors and Tc toxins, their N- and C-terminal extensions can differ significantly. The C-terminus of bacterial Rhs proteins encode for a diverse range of toxins and is often referred to as the hypervariable region (HVR) [ 31 ]. In Tc toxins, the C-terminal toxin is encapsulated within the Rhs cocoon [ 32 ], however, previous structural analyses of these proteins have been unable to resolve the structure of a HVR, suggesting that this region may be partially unfolded. So far, no high-resolution structural information exists for T6SS Rhs effectors.

In addition to its N-terminal prePAAR, TMD and PAAR regions, RhsA possesses Rhs elements, which are characterized by the presence of repeating tyrosine-aspartate (YD) motifs. The chromosomal regions encoding Rhs repeats were originally described in Escherichia coli genomes as sites where recombination frequently took place [ 17 ]. Later studies revealed that Rhs repeat-containing proteins are not unique to E. coli but are common across Proteobacteria [ 18 , 19 ]. Other more distant homologs, called teneurins, are involved in axon guidance in vertebrates [ 20 – 22 ]. Teneurins are type II single-pass transmembrane proteins that mediate cell-cell adhesion via their Rhs repeat-containing extracellular domains [ 23 , 24 ]. The intracellular domain consists of a transcriptional repressor that is released from the membrane upon proteolytic cleavage. The extracellular epidermal growth factor-like repeats (EGF) mediate covalent dimerization by formation of disulphide bridges [ 21 , 25 ]. Structures of bacterial Rhs proteins and the extracellular domain of human teneurin2 revealed that Rhs repeat regions form a large (~100 kDa) β-barrel cocoon-like structure spirally wound counterclockwise [ 26 – 29 ]. The C-terminus of the Rhs barrel is typically demarcated by a highly conserved PxxxxDPxGL motif, which is necessary for autoproteolytic cleavage [ 20 ]. Previous work has shown that C-terminal autoproteolysis of both T6SS Rhs effectors and toxin complex (Tc) Rhs toxins is required for toxin release from the Rhs cocoon [ 26 , 27 , 30 ].

(A) Genomic context of rhsA (PFL_6096, blue) in Pseudomonas protegens Pf-5. Upstream genes encoding the cognate VgrG protein, vgrG1 (PFL_6094, green) and RhsA-specific chaperone, eagR1 (PFL_6095, light brown) are shown. Self-protection against RhsA is conferred via expression of the downstream immunity-encoding gene, rhsI (dark brown). (B) Representative cryo-EM 2-D class averages of the assembled pre-firing complex composed of VgrG1, RhsA and EagR1 (left) and a schematic representation of each of the components that comprise this complex (right). Scale bar, 10 nm. (C) Full-length RhsA contains a prePAAR motif and a TMD comprising two transmembrane helices upstream of its PAAR domain. The RhsA ΔTMD truncation of RhsA was used in this study to determine the high-resolution structure of the RhsA cocoon. (D) Cryo-EM density of RhsA ΔTMD displayed perpendicular to the central symmetry axis of the barrel and rotated 90° clockwise (map postprocessed with DeepEMhancer). (E) Cartoon representation of the atomic model of RhsA colored in rainbow from N-terminus (blue) to C-terminus (red).

Previously, our group showed that the T6SS of the soil bacterium Pseudomonas protegens secretes the prePAAR and PAAR-containing effector RhsA [ 14 ]. The rhsA operon contains genes encoding its cognate VgrG spike and Eag chaperone proteins ( Fig 1A ), both of which are required for the delivery of this effector into susceptible competitors [ 14 ]. Previous negative stain electron microscopy experiments suggest that EagR1, RhsA and VgrG assemble to form a complex necessary for T6SS function ( Fig 1B ) [ 14 ]. The N-terminus of RhsA consists of a prePAAR motif as well as two predicted transmembrane helices within its TMD. The prePAAR motif is proposed to contribute sequence elements that ‘complete’ the PAAR domain and enable its interaction with the tip of VgrG1 [ 14 ] whereas the transmembrane helices likely play a role in target cell penetration [ 16 ].

Effectors are recruited to distinct regions of the T6SS tube-spike complex. Smaller effectors (< 50 kDa) are typically recognized and loaded within the lumen of the Hcp tube [ 12 ] whereas larger, multidomain effectors interact with VgrG proteins via an effector-encoded PAAR domain or by directly interacting with VgrG itself [ 13 ]. PAAR-containing effectors are widely distributed in Proteobacteria and can be further subdivided into subfamilies of proteins based on the presence of additional sequence motifs that may play a role in effector translocation into target cells. One family of PAAR effectors that was recently described in detail possess an additional N-terminal motif, called prePAAR, predicted to be involved in PAAR folding, as well as at least one N-terminal transmembrane domain (TMD) that is hypothesized to insert into the recipient cell inner membrane with one to three transmembrane helices per TMD that enable toxin translocation into the cytoplasm [ 14 ]. TMD-containing PAAR effectors require effector-associated gene (Eag) chaperones to bind and stabilize effector TMDs prior to export from the donor cell [ 14 , 15 ].

A functional T6SS apparatus requires the concerted action of three protein subcomplexes: a cell envelope-spanning membrane complex, a cytoplasmic baseplate complex and a bacteriophage tail-like sheath-tube complex [ 4 , 5 ]. The tail-like complex consists of a contractile sheath that surrounds an inner tube comprised of many copies of stacked ring-shaped hexameric hemolysin co-regulated protein (Hcp) [ 6 , 7 ]. This Hcp tube is capped with a member of the homotrimeric valine-glycine repeat protein G (VgrG) spike protein family, which typically also interacts with a cone-shaped proline-alanine-alanine-arginine (PAAR) domain-containing protein to form a complete T6SS tail tube-spike complex [ 8 ]. During a T6SS firing event, the Hcp-VgrG-PAAR tube-spike complex is rapidly assembled in the cytosol of the attacking bacterium, recruited to the membrane complex through its interaction with the baseplate, and subsequently surrounded by a contractile sheath that upon sheath contraction, propels the tail spike into the recipient cell [ 9 ]. Toxic effector proteins are recruited to the tube-spike complex and injected into recipient cells alongside Hcp and VgrG, typically resulting in death of the target cell. Self-protection from antibacterial T6SS effectors is accomplished via effector co-expression with cognate immunity proteins, which neutralize effector toxicity by occluding the effector active site or by the hydrolysis of effector generated toxic products [ 10 , 11 ].

One way that bacteria interact with their environment is by secreting toxic molecules into their surroundings. Many species of Gram-negative bacteria have evolved specialized protein secretion systems for this purpose with arguably the best-characterized example being the T6SS [ 1 , 2 ]. T6SSs mediate bacterial antagonism by facilitating the injection of toxic effector proteins into competing bacterial cells in a contact-dependent manner [ 3 ].

Results and discussion

RhsA possesses a unique plug domain at its N-terminus Our structural data show that the C-terminal aspartyl protease domain of RhsA seals the cocoon-shaped structure at one end, while the other end is capped by an N-terminal domain that adopts a smaller structure. This N-terminal domain of the barrel, formed by residues 268–386, caps the cocoon structure in a manner that is reminiscent of how a cork is used to plug a champagne bottle and thus we refer to it as the N-terminal plug domain (RhsA plug ) (Fig 3A–3E). The interface between the plug domain and the Rhs repeats is mainly stabilized by hydrophobic interactions (Fig 3A–3C) and a few hydrophilic interactions (S6A Fig). PPT PowerPoint slide

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TIFF original image Download: Fig 3. A unique plug domain seals the Rhs barrel of RhsA. (A) Surface representation of the cork domain of RhsA. The molecular surface is colored according to hydrophobicity where ochre and white indicate hydrophobic and hydrophilic regions, respectively. The Rhs barrel is shown as a cartoon. (B) Enlarged view of the cork domain of RhsA. The hydrophobic surface spirals around the domain as indicated by the black dashed line. (C) The upper Rhs repeats of RhsA possess complementary hydrophobic patches to those found on its plug domain (shown in cartoon representation, red). (D) The cocoon structure of RhsA is closed off by an N-terminal plug comprised of a ‘cork’ domain (orange, density representation), a ‘seal’ peptide (mesh), and an anchor helix (E, F). The seal and the cork density together form a cap structure. (E) The seal peptide, which also functions as the linker to the unmodelled PAAR domain, not only complements the shape of the cork but is also the entry point of the N-terminal part of the protein into the inside of the cocoon. The cocoon remains stably bound to the cleaved N-terminal region, including the PAAR domain, due to the anchor helix inside the cocoon. (F) The anchor helix of RhsA is stabilized by hydrophobic interactions with the inner wall of the Rhs repeats. (G) Cartoon representation of the cork region of the RhsA plug domain and a comparison with the homologous N-terminal plug domain of Photorhabdus luminescens TcdB2 (grey, PDB ID: 6H6G) and the unique FN-plug domain of human teneurin2 (green, PDB ID: 6FB3). The lower part of each depicted plug domain inserts into each of their respective Rhs barrels. The cork domain of RhsA is colored in rainbow. https://doi.org/10.1371/journal.ppat.1010182.g003 In addition to the cork structure (residues 305–386), the plug domain contains an anchor helix (residues 289–302) and an N-terminal seal peptide (residues 268–275). The seal peptide fills a small opening in the cork as it leaves the cocoon and connects the plug domain to the unmodelled PAAR domain (Figs 3D–3E and S7A and S7B). In doing so, the seal peptide, together with the cork, closes off the cocoon entirely (S7B Fig). The anchor helix is amphipathic and stabilized by interactions with a hydrophobic patch of the Rhs repeats in this region (Figs 3E and 3F and S7C–S7E). The position of the anchor helix in our structure suggests its function is twofold. On the one hand it holds the N-terminal cleavage site in place. On the other hand, it ensures that the N-terminal plug domain remains stably attached to the cocoon, so that it remains sealed even after N-terminal cleavage. The plug domain of RhsA possesses sequence and structural similarity to a domain found in the BC components of TcdB2-TccC3 [26] and YenBC [27] (Figs 3G and S8A–S8C) (18% sequence identity to TcdB2; 22% sequence identity to YenB). Interestingly, in Tc toxins this domain does not act as a plug that prevents the release of the toxin, but instead forms a negatively charged constriction through which the toxin domain is threaded prior to its translocation into a target cell [32]. A plug domain has also been described in teneurins (called fibronectin-plug, FN-plug) [28,29], however, it does not bear sequence or structural similarity to the plug domain of RhsA described herein. The FN-plug is narrower than the plug domains of RhsA and Tc toxins and extends further into the Rhs cocoon structure (Fig 3G) making numerous hydrophilic interactions with residues lining the inside of the YD-shell [28]. Our structure suggests that in contrast to Tc toxins the plug domain of RhsA tightly seals its Rhs cocoon. Because the plug domain appears to strongly interact with the Rhs barrel, mechanical removal of this entire domain after translocation of the cocoon into the target cell cytosol seems unlikely. Instead, we propose that only the N-terminal peptide seal of the RhsA plug is pulled out during T6SS-dependent delivery of RhsA into a susceptible bacterial cell. The seal peptide is part of the N-terminal region that is proteolytically cleaved, so we speculate that it would be more easily removed compared to the entire plug domain, which is held in place by the anchor helix that exists downstream of the N-terminal cleavage site. Penetration of the outer membrane and peptidoglycan layer as well as translocation of RhsA through the target cell inner membrane could provide the mechanical force to remove the seal peptide, creating a channel through which the unfolded toxin domain could exit. Since the plug domain resembles the constriction site in Tc toxins that effectors must pass during the initial translocation process, the same process is conceivable for the release of the C-terminal toxin domain of RhsA.

Comparison between Rhs proteins of known structure A common feature of all Rhs proteins that have been structurally characterized to date is the cocoon structure formed by the Rhs repeats. While the Rhs cocoons from RhsA and teneurin2 are comprised of three β-helical turns of Rhs repeats, the cocoons of Tc toxins have four turns and therefore have bigger overall dimensions and an internal cavity with larger volume. Consistent with this observation, the effector domain inside Tc toxin cocoons is larger (~30 kDa) than the toxin domain of RhsA (~15 kDa). All characterized Rhs proteins contain a conserved C-terminal region. For Tc toxins and RhsA this domain functions as aspartyl autoprotease whereas in teneurins it acts as a YD-shell plug that is not cleaved (Fig 4 blue). In all instances, the C-terminal conserved region comprises 14 Rhs repeats that spiral into the inside of the cocoon and seal it at one end. PPT PowerPoint slide

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TIFF original image Download: Fig 4. Structural comparison of Rhs repeat containing proteins. The C-terminal autoproteolysis domain (left and middle) or YD-shell plug (right) is structurally conserved among diverse Rhs proteins (blue). RhsA and BC components of Tc toxin complexes encapsulate their toxic effector domains (red) whereas in teneurin proteins the toxin domain is appended to the outside of the Rhs barrel (red). A distinguishing feature of these Rhs proteins is the unique N-terminal plug domain for each protein family (orange). RhsA is capped by a cork-like plug domain that seals the Rhs barrel (orange). In Tc toxins, the homologous plug domain acts as constriction site and the cocoon is sealed off by a β-propeller domain (green). Teneurin proteins are capped with a non-homologous FN-plug (orange) that is stabilized by an NHL domain (green). The lower row shows schematic representations of the domain organizations. https://doi.org/10.1371/journal.ppat.1010182.g004 The plug domains that close off the N-terminal end of the cocoon structures are more variable than their C-terminal counterparts (Fig 4 orange). While the barrel of teneurins is sealed with an FN-plug that is mainly held in place by hydrophilic interactions with the interior of the barrel, TcdB2 from Photorhabdus luminescens and other BC components of Tc toxins close the cocoon using a β-propeller domain that acts as gatekeeper for toxin release. RhsA uses an N-terminal plug domain, which is homologous to the domain that forms the constriction site in Tc toxins. Both TcBC and RhsA encapsule a toxic effector, whereas teneurins act as scaffolding proteins with an empty cocoon (Fig 4 red). Nevertheless, teneurins encode a C-terminal ancient toxin component that sits outside of the barrel and is inactive (ABD Tox-GHH, Fig 4 red). While RhsA contains two autoprotease sites, namely an aspartyl protease that is responsible for the cleavage of the C-terminal toxic effector and an unknown protease site that cleaves the N-terminal 304 residues of the protein, Tc toxins only contain the former site and teneurins none at all. In conclusion, based on its unique plug domain and its fusion to an N-terminal PAAR domain, we propose that our RhsA structure represents the founding member of a third structural class of Rhs repeat containing proteins (Fig 4).

Architecture of the pre-firing complex To investigate how RhsA is mounted onto VgrG1 we set out to determine the structure of the secretion competent pre-firing complex (PFC) comprising VgrG1 in complex with full-length RhsA and EagR1. We purified the complex as described previously [14] and examined it by single particle cryo-EM. The 2D class averages enabled us to unequivocally characterize the arrangement of the subunits within the complex (Fig 5A). Expectedly, both EagR1 and RhsA are located at the tip of VgrG1. Interestingly, a large fraction of PFCs contained RhsA dimers instead of monomers (S9B Fig), similar to what was observed in our analysis of RhsA ΔTMD alone. This demonstrates that the loading of two RhsA molecules onto one VgrG1 is sterically possible although it is unlikely to occur in vivo given that current data indicates a single VgrG homotrimer caps the Hcp tube [36]. PPT PowerPoint slide

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TIFF original image Download: Fig 5. High-resolution structure of P. protegens VgrG1. (A) The RhsA barrel of the assembled pre-firing complex (PFC) displays high positional flexibility relative to VgrG1. Representative cryo-EM 2-D class averages depicting flexibility are shown. Scale bar, 10 nm. (B) Cryo-EM density and (C) ribbon representation of the molecular model of the P. protegens VgrG1 trimer viewed along the long axis of the protein. Each protomer is colored differently to highlight their positions within the homotrimeric VgrG1 spike. https://doi.org/10.1371/journal.ppat.1010182.g005 As is the case for the previously characterized VgrG1-EagT6-Tse6 complex from P. aeruginosa [16], T6SS effectors can adopt multiple positions relative to their cognate VgrG spike protein. Unfortunately, this conformational heterogeneity prevented us from obtaining a high-resolution 3D reconstruction of the entire complex (S1 Movie). Instead, we applied C 3 symmetry during processing to determine the three-dimensional structure of P. protegens VgrG1 (Fig 5B). In applying this symmetry operator, the RhsA and EagR1 components of the complex, which do not adopt this symmetry, are averaged out during image processing. The cryo-EM map of VgrG1 reached a resolution of 3.3 Å and allowed us to build almost the complete atomic model of the protein comprising residues 8–643 (Fig 5C and Table 1). As expected, given its high sequence homology, the structure of VgrG1 from P. protegens is nearly identical (71% sequence identity, 81% sequence similarity) to our previously determined VgrG1 structure from P. aeruginosa (r.m.s.d of 1.035 between 544 pruned Cα atoms; r.m.s.d of 1.428 across the complete structure, S9B and S9C Fig) [16] even though their respective effectors, RhsA and Tse6, bear no sequence or structural similarity to one another beyond their N-terminal prePAAR and PAAR domains. Intriguingly, we identified two spherical densities in the center of the β-sheet prism of the VgrG1 trimer (S9E Fig). Since we also observed these densities previously in the VgrG1 structure from P. aeruginosa [16], we speculate that this may be a common feature of VgrG1 proteins. Based on the exclusive clustering of positively charged residues around this density and its overall size, we hypothesize that it corresponds to either a phosphate or sulfate anion, which probably helps to stabilize the core of VgrG1 given that in the absence of this anion, the presence of positively charged residues in the core of the protein would be energetically unfavorable.

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[1] Url: https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1010182

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