Yannick R. Brunet

Structure and Regulation of the Type VI Secretion System

The type VI secretion system (T6SS) is a complex and widespread gram-negative bacterial export pathway with the capacity to translocate protein effectors into a diversity of target cell types. Current structural models of the T6SS indicate that the apparatus is composed of at least two complexes, a dynamic bacteriophage-like structure and a cell-envelope-spanning membrane-associated assembly. How these complexes interact to promote effector secretion and cell targeting remains a major question in the field. As a contact-dependent pathway with specific cellular targets, the T6SS is subject to tight regulation. Thus, the identification of regulatory elements that control T6S expression continues to shape our understanding of the environmental circumstances relevant to its function. This review discusses recent progress toward characterizing T6S structure and regulation.

Nooks and Crannies in Type VI Secretion Regulation

Type VI secretion systems (T6SS) are macromolecular, transenvelope machines encoded within the genomes of most Gram-negative bacteria, including plant, animal, and human pathogens, as well as soil and environmental isolates. T6SS are involved in a broad variety of functions: from pathogenesis to biofilm formation and stress sensing. This large array of functions is reflected by a vast diversity of regulatory mechanisms: repression by histone-like proteins and regulation by quorum sensing, transcriptional factors, two-component systems, alternative sigma factors, or small regulatory RNAs. Finally, T6SS may be produced in an inactive state and are turned on through the action of a posttranslational cascade involving phosphorylation and subunit recruitment. The current data reviewed here highlight how T6SS have been integrated into existing regulatory networks and how the expression of the T6SS loci is precisely modulated to adapt T6SS production to the specific needs of individual bacteria.

Imaging Type VI Secretion-Mediated Bacterial Killing

In the environment, bacteria compete with each other for nutrient availability or to extend their ecological niche. The type VI secretion system contributes to bacterial competition by the translocation of antibacterial effectors from predators into prey cells. The T6SS assembles a dynamic structure-the sheath-wrapped around a tube constituted of the Hcp protein. It has been proposed that by cycling between extended and contracted conformations the sheath acts as a crossbow to propel the Hcp tube toward the target cell. While the sheath dynamics have been studied in monocultures, the activity of the T6SS has not been recorded in presence of the prey. Here, time-lapse fluorescence microscopy of cocultures demonstrates that prey cells are killed upon contact with predator cells. Additional experiments provide evidence that sheath contraction correlates with nearby cell fading and that prey lysis occurs within minutes after sheath contraction. The results support a model in which T6SS dynamics are responsible for T6SS effectors translocation into recipient cells.

Type VI secretion and bacteriophage tail tubes share a common assembly pathway

The Type VI secretion system (T6SS) is a widespread macromolecular structure that delivers protein effectors to both eukaryotic and prokaryotic recipient cells. The current model describes the T6SS as an inverted phage tail composed of a sheath‐like structure wrapped around a tube assembled by stacked Hcp hexamers. Although recent progress has been made to understand T6SS sheath assembly and dynamics, there is no evidence that Hcp forms tubes in vivo. Here we show that Hcp interacts with TssB, a component of the T6SS sheath. Using a cysteine substitution approach, we demonstrate that Hcp hexamers assemble tubes in an ordered manner with a head‐to‐tail stacking that are used as a scaffold for polymerization of the TssB/C sheath‐like structure. Finally, we show that VgrG but not TssB/C controls the proper assembly of the Hcp tubular structure. These results highlight the conservation in the assembly mechanisms between the T6SS and the bacteriophage tail tube/sheath.

TssK Is a Trimeric Cytoplasmic Protein Interacting with Components of Both Phage-like and Membrane Anchoring Complexes of the Type VI Secretion System

Background: The T6SS assembles from 13 proteins that form two sub-assemblies. Results: TssK is a cytoplasmic protein that interacts with Hcp, TssC, TssL, and TssA. Conclusion: The TssK complex is three-arm shaped and links the membrane and phage-like complexes in T6SS. Significance: The structural and functional characterization of TssK leads to a better understanding of T6SS assembly. The Type VI secretion system (T6SS) is a macromolecular machine that mediates bacteria-host or bacteria-bacteria interactions. The T6SS core apparatus assembles from 13 proteins that form two sub-assemblies: a phage-like complex and a trans-envelope complex. The Hcp, VgrG, TssE, and TssB/C subunits are structurally and functionally related to components of the tail of contractile bacteriophages. This phage-like structure is thought to be anchored to the membrane by a trans-envelope complex composed of the TssJ, TssL, and TssM proteins. However, how the two sub-complexes are connected remains unknown. Here we identify TssK, a protein that establishes contacts with the two T6SS sub-complexes through direct interactions with TssL, Hcp, and TssC. TssK is a cytoplasmic protein assembling trimers that display a three-armed shape, as revealed by TEM and SAXS analyses. Fluorescence microscopy experiments further demonstrate the requirement of TssK for sheath assembly. Our results suggest a central role for TssK by linking both complexes during T6SS assembly.

An Epigenetic Switch Involving Overlapping Fur and DNA Methylation Optimizes Expression of a Type VI Secretion Gene Cluster

Type VI secretion systems (T6SS) are macromolecular machines of the cell envelope of Gram-negative bacteria responsible for bacterial killing and/or virulence towards different host cells. Here, we characterized the regulatory mechanism underlying expression of the enteroagregative Escherichia coli sci1 T6SS gene cluster. We identified Fur as the main regulator of the sci1 cluster. A detailed analysis of the promoter region showed the presence of three GATC motifs, which are target of the DNA adenine methylase Dam. Using a combination of reporter fusion, gel shift, and in vivo and in vitro Dam methylation assays, we dissected the regulatory role of Fur and Dam-dependent methylation. We showed that the sci1 gene cluster expression is under the control of an epigenetic switch depending on methylation: fur binding prevents methylation of a GATC motif, whereas methylation at this specific site decreases the affinity of Fur for its binding box. A model is proposed in which the sci1 promoter is regulated by iron availability, adenine methylation, and DNA replication.

The Type VI Secretion TssEFGK-VgrG Phage-Like Baseplate Is Recruited to the TssJLM Membrane Complex via Multiple Contacts and Serves As Assembly Platform for Tail Tube/Sheath Polymerization.

The Type VI secretion system (T6SS) is a widespread weapon dedicated to the delivery of toxin proteins into eukaryotic and prokaryotic cells. The 13 T6SS subunits assemble a cytoplasmic contractile structure anchored to the cell envelope by a membrane-spanning complex. This structure is evolutionarily, structurally and functionally related to the tail of contractile bacteriophages. In bacteriophages, the tail assembles onto a protein complex, referred to as the baseplate, that not only serves as a platform during assembly of the tube and sheath, but also triggers the contraction of the sheath. Although progress has been made in understanding T6SS assembly and function, the composition of the T6SS baseplate remains mostly unknown. Here, we report that six T6SS proteins–TssA, TssE, TssF, TssG, TssK and VgrG–are required for proper assembly of the T6SS tail tube, and a complex between VgrG, TssE,-F and-G could be isolated. In addition, we demonstrate that TssF and TssG share limited sequence homologies with known phage components, and we report the interaction network between these subunits and other baseplate and tail components. In agreement with the baseplate being the assembly platform for the tail, fluorescence microscopy analyses of functional GFP-TssF and TssK-GFP fusion proteins show that these proteins assemble stable and static clusters on which the sheath polymerizes. Finally, we show that recruitment of the baseplate to the apparatus requires initial positioning of the membrane complex and contacts between TssG and the inner membrane TssM protein.

Priming and polymerization of a bacterial contractile tail structure

Contractile tails are composed of an inner tube wrapped by an outer sheath assembled in an extended, metastable conformation that stores mechanical energy necessary for its contraction. Contraction is used to propel the rigid inner tube towards target cells for DNA or toxin delivery. Although recent studies have revealed the structure of the contractile sheath of the type VI secretion system, the mechanisms by which its polymerization is controlled and coordinated with the assembly of the inner tube remain unknown. Here we show that the starfish-like TssA dodecameric complex interacts with tube and sheath components. Fluorescence microscopy experiments in enteroaggregative Escherichia coli reveal that TssA binds first to the type VI secretion system membrane core complex and then initiates tail polymerization. TssA remains at the tip of the growing structure and incorporates new tube and sheath blocks. On the basis of these results, we propose that TssA primes and coordinates tail tube and sheath biogenesis.

Regulation of Type VI Secretion Gene Clusters by σ54 and Cognate Enhancer Binding Proteins

Type VI secretion systems (T6SS) are bacteriophage-derived macromolecular machines responsible for the release of at least two proteins in the milieu, which are thought to form an extracellular appendage. Although several T6SS have been shown to be involved in the virulence of animal and plant pathogens, clusters encoding these machines are found in the genomes of most species of gram-negative bacteria, including soil, marine, and environmental isolates. T6SS have been associated with several phenotypes, ranging from virulence to biofilm formation or stress sensing. Their various environmental niches and large diversity of functions are correlated with their broad variety of regulatory mechanisms. Using a bioinformatic approach, we identified several clusters, including those of Vibrio cholerae, Aeromonas hydrophila, Pectobacterium atrosepticum, Pseudomonas aeruginosa, Pseudomonas syringae pv. tomato, and a Marinomonas sp., which possess typical -24/-12 sequences, recognized by the alternate sigma factor sigma 54 (σ(54) or σ(N)). σ(54), which directs the RNA polymerase to these promoters, requires the action of a bacterial enhancer binding protein (bEBP), which binds to cis-acting upstream activating sequences. Putative bEBPs are encoded within the T6SS gene clusters possessing σ(54) boxes. Using in vitro binding experiments and in vivo reporter fusion assays, we showed that the expression of these clusters is dependent on both σ(54) and bEBPs.

Crystal Structure and Self-Interaction of the Type VI Secretion Tail-Tube Protein from Enteroaggregative Escherichia coli

The type VI secretion system (T6SS) is a widespread machine used by bacteria to control their environment and kill or disable bacterial species or eukaryotes through toxin injection. The T6SS comprises a central tube formed of stacked hexamers of hemolysin co-regulated proteins (Hcp) and terminated by a trimeric valine-glycine repeat protein G (VgrG) component, the cell puncturing device. A contractile tail sheath, formed by the TssB and TssC proteins, surrounds this tube. This syringe-like machine has been compared to an inverted phage, as both Hcp and VgrG share structural homology with tail components of Caudovirales. Here we solved the crystal structure of a tryptophan-substituted double mutant of Hcp1 from enteroaggregative Escherichia coli and compared it to the structures of other Hcps. Interestingly, we observed that the purified Hcp native protein is unable to form tubes in vitro. To better understand the rationale for observation, we measured the affinity of Hcp1 hexamers with themselves by surface plasmon resonance. The intra-hexamer interaction is weak, with a KD value of 7.2 µM. However, by engineering double cysteine mutants at defined positions, tubes of Hcp1 gathering up to 15 stacked hexamers formed in oxidative conditions. These results, together with those available in the literature regarding TssB and TssC, suggest that assembly of the T6SS tube differs significantly from that of Sipho- or Myoviridae.