Crystal structure and self-interaction of the type VI secretion tail-tube protein from enteroaggregative Escherichia coli (original) (raw)
Related papers
Scientific reports, 2016
The Type VI secretion system (T6SS) is a versatile machine that delivers toxins into either eukaryotic or bacterial cells. It thus represents a key player in bacterial pathogenesis and inter-bacterial competition. Schematically, the T6SS can be viewed as a contractile tail structure anchored to the cell envelope. The contraction of the tail sheath propels the inner tube loaded with effectors towards the target cell. The components of the contracted tail sheath are then recycled by the ClpV AAA(+) ATPase for a new cycle of tail elongation. The T6SS is widespread in Gram-negative bacteria and most of their genomes carry several copies of T6SS gene clusters, which might be activated in different conditions. Here, we show that the ClpV ATPases encoded within the two T6SS gene clusters of enteroaggregative Escherichia coli are not interchangeable and specifically participate to the activity of their cognate T6SS. Here we show that this specificity is dictated by interaction between the C...
PLOS Pathogens, 2011
Type VI secretion systems (T6SS) are trans-envelope machines dedicated to the secretion of virulence factors into eukaryotic or prokaryotic cells, therefore required for pathogenesis and/or for competition towards neighboring bacteria. The T6SS apparatus resembles the injection device of bacteriophage T4, and is anchored to the cell envelope through a membrane complex. This membrane complex is composed of the TssL, TssM and TagL inner membrane anchored proteins and of the TssJ outer membrane lipoprotein. Here, we report the crystal structure of the enteroaggregative Escherichia coli Sci1 TssJ lipoprotein, a two four-stranded b-sheets protein that exhibits a transthyretin fold with an additional a-helical domain and a protruding loop. We showed that TssJ contacts TssM through this loop since a loop depleted mutant failed to interact with TssM in vitro or in vivo. Biophysical analysis of TssM and TssJ-TssM interaction suggest a structural model of the membraneanchored outer shell of T6SS. Collectively, our results provide an improved understanding of T6SS assembly and encourage structure-aided drug design of novel antimicrobials targeting T6SS.
Biogenesis and structure of a type VI secretion membrane core complex
Nature, 2015
Bacteria share their ecological niches with other microbes. The bacterial Type VI secretion system is one of the key players for microbial competition, as well as an important virulence determinant during bacterial infections. It assembles a nanocrossbow-like structure that propels an arrow made of Hcp tube and VgrG spike into the cytoplasm of the attacker cell and punctures the prey's cell wall. The nano-crossbow is stably anchored to the cell envelope of the attacker by a membrane core complex. Here, we show that this complex is assembled by the sequential addition of three proteins-TssJ, TssM and TssL-and present a 11.6 Å resolution structure of the fully assembled complex, determined by negative stain electron microscopy. With overall C5 symmetry, this 1.7-megadalton complex comprises a large base in the cytoplasm. It extends in the periplasm via 10 arches to form a double-ring structure containing the Cterminal domain of TssM (TssM ct) and TssJ that is anchored in the outer membrane. The crystal structure of the TssM ct-TssJ complex coupled to whole-cell accessibility studies suggest that large conformational changes induce transient pore formation in the outer membrane allowing passage of the attacking Hcp tube/VgrG spike.
Nature Microbiology, 2017
The Type VI secretion system (T6SS) is a multiprotein machine widespread in Gram-negative bacteria that delivers toxins into both eukaryotic and prokaryotic cells. The mechanism of action of the T6SS is comparable to that of contractile myophages. The T6SS builds a tail-like structure made of an inner tube wrapped by a sheath assembled under an extended conformation. Contraction of the sheath propels the inner tube toward the target cell. The T6SS tail is assembled on a platform-the baseplatefunctionally similar to bacteriophage baseplates. In addition, the baseplate docks the tail to a trans-envelope membrane complex that orients the tail toward the target. Here, we report the crystal structure of TssK, a central component of the T6SS baseplate. We show that TssK is constituted of three domains and establish the contribution of each domain to the interaction with TssK partners. Importantly, this study reveals that the N-terminal domain of TssK is structurally homologous to the shoulder domain of phage receptor binding proteins while the C-terminal domain binds the membrane complex. We propose that TssK has conserved the domain of attachment to the virion particle but has evolved the reception domain to use the T6SS membrane complex as receptor.
Biogenesis and structure of a type VI secretion baseplate
Nature Microbiology
To support their growth in a competitive environment and cause pathogenesis, bacteria have evolved a broad repertoire of macromolecular machineries to deliver specific effectors and toxins. Among these multiprotein complexes, the type VI secretion system (T6SS) is a contractile nanomachine that targets both prokaryotic and eukaryotic cells. The T6SS comprises two functional sub-complexes: a bacteriophage-related tail structure anchored to the cell envelope by a membrane complex. As in other contractile injection systems, the tail is composed of an inner tube wrapped by a sheath and built on the baseplate. In the T6SS, the baseplate is not only the tail assembly platform, but also docks the tail to the membrane complex and hence serves as an evolutionary adaptor. Here we define the biogenesis pathway and report the cryo-EM structure the wedge protein complex of the T6SS from Enteroaggregative Escherichia coli (EAEC). Using an integrative approach, we unveil the molecular architecture of the whole T6SS baseplate and its interaction with the tail sheath, offering detailed insights into its biogenesis and function. We discuss architectural and mechanistic similarities but also revealed key differences with the T4 phage and Mu phage baseplates. INTRODUCTION The bacterial Type VI secretion system (T6SS) is one of the key players for microbial competition, and an important virulence factor during bacterial infections. This versatile nanomachine delivers a wide arsenal of effector proteins directly into prokaryotic and eukaryotic target cells 1-4. T6SS anti-bacterial activities promote privileged access to the niche, to nutrients or to DNA. In most cases, T6SS causes damage within competitor bacterial cells and therefore participates in the reshaping of bacterial communities such as the Native PAGE profiles immunodetected with anti-GFP antibodies revealed the presence of a high-molecular weight complex (HMWC) with a size of ~ 800 kDa (Fig. 1b). This complex does not contain TssE, TssA, VgrG and TssM and likely corresponds to TssK sfGFP FG since (1) it disappears in the absence of tssF or tssG, (2) a HMWC of a comparable size is observable upon pull-down of TssK sfGFP co-produced with TssF and TssG in the heterologous host E. coli BL21(DE3), and (3) analysis of this HMWC on denaturing SDS-PAGE reveals the presence of TssK sfGFP , TssF and TssG (Fig. 1b). Taken together, the fluorescence microscopy and native-PAGE results, and the previous reports of TssKFG and TssKFGE complex purifications in Serratia marcescens and E. coli 36,37 , suggest that the TssKFG complex represents a stable intermediate during T6SS baseplate biogenesis. We therefore propose that T6SS baseplate biogenesis starts with the formation of the TssKFG complex and then proceeds with the polymerization of TssKFG building units around the VgrG hub. The observation that TssE is not required for TssKFG-VgrG complex formation, further suggests that TssE binds to the TssKFG either prior to or after its polymerization. This assembly pathway is comparable to that of the minimal phage baseplate, in which gp25 attaches to the baseplate either after completion of the gp10-7-8-6-53 complex 40 or at a later stage, triggering the polymerization of the contractile sheath 41. Interaction network within the T6SS baseplate To gain further insight into the connectivity network between the T6SS baseplate components, we performed a systematic biochemical pull-down assay (Supplementary Fig. 2a-e). This approach confirmed or revealed a number of contacts including interactions between TssG and TssF, TssE, and TssK (Fig. 1c). We then tested whether intermediate subcomplexes, suggested by the assembly pathway defined above, could be purified. In agreement with the proposed assembly pathway, we succeeded to pull-down biogenesis intermediate complexes consisting of TssFG, TssKFG and TssKFGE (Supplementary Fig. 2a-b). Based on these data, we propose that the TssKFGE sub-complex represents the T6SS equivalent of the bacteriophage wedge unit (TssFGE), bound to the TssK membrane complex adaptor. Purification, stoichiometry and cryo-EM structure of a T6SS wedge complex
Structural biology of type VI secretion systems
Philosophical transactions of the Royal Society of London. Series B, Biological sciences, 2012
Type VI secretion systems (T6SSs) are transenvelope complexes specialized in the transport of proteins or domains directly into target cells. These systems are versatile as they can target either eukaryotic host cells and therefore modulate the bacteria-host interaction and pathogenesis or bacterial cells and therefore facilitate access to a specific niche. These molecular machines comprise at least 13 proteins. Although recent years have witnessed advances in the role and function of these secretion systems, little is known about how these complexes assemble in the cell envelope. Interestingly, the current information converges to the idea that T6SSs are composed of two subassemblies, one resembling the contractile bacteriophage tail, whereas the other subunits are embedded in the inner and outer membranes and anchor the bacteriophage-like structure to the cell envelope. In this review, we summarize recent structural information on individual T6SS components emphasizing the fact th...
The Journal of Biochemistry, 2013
The bacterial Type 6 secretion system (T6SS) translocates protein toxins (also called effectors) from the cytosol of a T6SS-carrying cell to a target cell by a syringe-like supramolecular complex resembling a contractile tail of bacteriophages. Valine-glycine repeat protein G (VgrG) proteins, which are the homologues of the gp27-gp5 (gene product) cell puncturing complex of bacteriophage T4, are considered to be located at the attacking tip of the bacterial T6SS apparatus. Here, we over-expressed six VgrG proteins from pathogenic Escherichia coli O157 and CFT073 strains. Purified VgrG1 of E. coli O157 and c3393 of E. coli CFT073 form trimer in solution and are rich in b-structure. We also solved the crystal structure of a trypsin-resistant C-terminal fragment of E. coli O157 VgrG1 (VgrG1C G561) at 1.95 Å resolution. VgrG1C G561 forms a three-stranded antiparallel b-helix which is structurally similar to the b-helix domain of the central spike protein (gp138) of phi92 phage, indicating a possible evolutional relationship. Comparison of four different three-stranded b-helix proteins shows how their amino acid composition determines the protein fold.
Novel structural components generate distinct type VI secretion system anchoring modes
2020
ABSTRACTThe type VI secretion system (T6SS) is a phage-derived contractile nanomachine primarily involved in interbacterial competition. Its pivotal component, TssA, is indispensable for the assembly of the T6SS sheath structure, the contraction of which propels a payload of effector proteins into neighboring cells. Despite their key function, TssA proteins exhibit unexpected diversity and exist in two major forms, a short (TssAS) and a long (TssAL) TssA. Whilst TssALproteins interact with a partner, called TagA, to anchor the distal end of the extended sheath, the mechanism for the stabilization of TssAS-containing T6SSs remains unknown. Here we discover a novel class of structural components that interact with short TssA proteins and contribute to T6SS assembly by stabilizing the polymerizing sheath from the baseplate. We demonstrate that the presence of these components is important for full sheath extension and optimal firing. Moreover, we show that the pairing of each form of T...
Frontiers in Microbiology
Bacterial pathogens utilize a myriad of mechanisms to invade mammalian hosts, damage tissue sites, and evade the immune system. One essential strategy of Gramnegative bacteria is the secretion of virulence factors through both inner and outer membranes to reach a potential target. Most secretion systems are harbored in mobile elements including transposons, plasmids, pathogenicity islands, and phages, and Escherichia coli is one of the more versatile bacteria adopting this genetic information by horizontal gene transfer. Additionally, E. coli is a bacterial species with members of the commensal intestinal microbiota and pathogens associated with numerous types of infections such as intestinal, urinary, and systemic in humans and other animals. T6SS cluster plasticity suggests evolutionarily divergent systems were acquired horizontally. T6SS is a secretion nanomachine that is extended through the bacterial double membrane; from this apparatus, substrates are conveyed straight from the cytoplasm of the bacterium into a target cell or to the extracellular space. This nanomachine consists of three main complexes: proteins in the inner membrane that are T4SS component-like, the baseplate complex, and the tail complex, which are formed by components evolutionarily related to contractile bacteriophage tails. Advances in the T6SS understanding include the functional and structural characterization of at least 13 subunits (so-called core components), which are thought to comprise the minimal apparatus. So far, the main role of T6SS is on bacterial competition by using it to kill neighboring non-immune bacteria for which antibacterial proteins are secreted directly into the periplasm of the bacterial target after cell-cell contact. Interestingly, a few T6SSs have been associated directly to pathogenesis, e.g., roles in biofilm formation and macrophage survival. Here, we focus on the advances on T6SS from the perspective of E. coli pathotypes with emphasis in the secretion apparatus architecture, the mechanisms of pathogenicity of effector proteins, and the events of lateral gene transfer that led to its spread.
TssA forms a gp6-like ring attached to the type VI secretion sheath
The EMBO journal, 2016
The type VI secretion system (T6SS) is a supra-molecular bacterial complex that resembles phage tails. It is a killing machine which fires toxins into target cells upon contraction of its TssBC sheath. Here, we show that TssA1 is a T6SS component forming dodecameric ring structures whose dimensions match those of the TssBC sheath and which can accommodate the inner Hcp tube. The TssA1 ring complex binds the T6SS sheath and impacts its behaviour in vivo In the phage, the first disc of the gp18 sheath sits on a baseplate wherein gp6 is a dodecameric ring. We found remarkable sequence and structural similarities between TssA1 and gp6 C-termini, and propose that TssA1 could be a baseplate component of the T6SS Furthermore, we identified similarities between TssK1 and gp8, the former interacting with TssA1 while the latter is found in the outer radius of the gp6 ring. These observations, combined with similarities between TssF and gp6N-terminus or TssG and gp53, lead us to propose a comp...