In situ and high‐resolution cryo‐EM structure of a bacterial type VI secretion system membrane complex (original) (raw)
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In situ and high-resolution Cryo-EM structure of the Type VI secretion membrane complex
Bacteria have evolved macromolecular machineries that secrete effectors and toxins to survive and thrive in diverse environments. The type VI secretion system (T6SS) is a contractile machine that is composed of a baseplate that contains a spike onto which an inner tube is built, surrounded by a contractile sheath. The T6SS is an intracellular machine inserted in the bacterial membranes by a membrane complex. This membrane complex (MC) comprises three proteins: TssJ, TssL and TssM. We previously reported the low-resolution negative stain electron microscopy structure of the enteroaggregative Escherichia coli MC and proposed a rotational 5-fold symmetry with a TssJ:TssL:TssM stoichiometry of 2:2:2. Here, cryo-electron tomography and single particle analysis CryoEM of the T6SS MC confirmed the 5-fold symmetry in situ and identified the regions of the structure that insert into the bacterial membranes. A high resolution model obtained by reveals its global architecture and highlights ne...
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.
Structure of a Type IV Secretion System Core Complex
Science, 2009
Type IV secretion systems (T4SSs) are important virulence factors used by Gram-negative bacterial pathogens to inject effectors into host cells or to spread plasmids harboring antibiotic resistance genes. We report the 15 angstrom resolution cryo–electron microscopy structure of the core complex of a T4SS. The core complex is composed of three proteins, each present in 14 copies and forming a ∼1.1-megadalton two-chambered, double membrane–spanning channel. The structure is double-walled, with each component apparently spanning a large part of the channel. The complex is open on the cytoplasmic side and constricted on the extracellular side. Overall, the T4SS core complex structure is different in both architecture and composition from the other known double membrane–spanning secretion system that has been structurally characterized.
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
F1000 - Post-publication peer review of the biomedical literature, 2004
Type III secretion systems (TTSSs) mediate translocation of virulence factors into host cells. We report the 17-angstrom resolution structures of a central component of Salmonella typhimurium TTSS, the needle complex, and its assembly precursor, the bacterial envelope-anchored base. Both the base and the fully assembled needle complex adopted multiple oligomeric states in vivo, and needle assembly was accompanied by recruitment of the protein PrgJ as a structural component of the base. Moreover, conformational changes during needle assembly created scaffolds for anchoring both PrgJ and the needle substructure and may provide the basis for substrate-specificity switching during type III secretion. Type III secretion systems (TTSSs) are central to the virulence of many Gram-negative bacteria pathogenic for animals and plants (1, 2). In addition to the needle complex (3), which is the core component of these systems, TTSSs are composed of more than 20 proteins, including a highly conserved group of integral membrane proteins, a family of customized cytoplasmic chaperones, and several accessory proteins (1, 2), placing TTSSs among the most complex protein secretion systems known. In S. typhimurium, the needle complex is formed by a base and a filamentous needle, composed of a single protein, PrgI, that projects ~50 nm from the bacterial surface (Fig. 1, A and B) (3). The base is formed by InvG, PrgH, and PrgK (4) and features four distinct rings, two associated with the outer membrane (OR1 and OR2 in Fig. 1A) and another two that are in close proximity to the inner membrane (IR1 and IR2 in Fig. 1A). The entire complex is essential for virulence (5) and is believed to provide a conduit for the direct transport of proteins from the bacterial cytoplasm to the host cell. Here, we have used electron cryomicroscopy to visualize the detailed structural organization of the S. typhimurium needle complex, as well as structural changes that occur during the last step of its assembly.
Near-atomic-resolution cryo-EM analysis of the Salmonella T3S injectisome basal body
The type III secretion (T3S) injectisome is a specialized protein nanomachine that is critical for the pathogenicity of many Gram-negative bacteria, including purveyors of plague, typhoid fever, whooping cough, sexually transmitted infections and major nosocomial infections. This syringe-shaped 3.5-MDa macromolecular assembly spans both bacterial membranes and that of the infected host cell. The internal channel formed by the injectisome allows for the direct delivery of partially unfolded virulence effectors into the host cytoplasm 1. The structural foundation of the injectisome is the basal body, a molecular lock-nut structure composed predominantly of three proteins that form highly oligomerized concentric rings spanning the inner and outer membranes 2–5. Here we present the structure of the prototypical Salmonella enterica serovar Typhimurium pathogenicity island 1 basal body, determined using single-particle cryo-electron microscopy, with the inner-membrane-ring and outer-membrane-ring oligomers defined at 4.3 Å and 3.6 Å resolution, respectively. This work presents the first, to our knowledge, high-resolution structural characterization of the major components of the basal body in the assembled state, including that of the widespread class of outer-membrane portals known as secretins.
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...
Structure of the outer membrane complex of a type IV secretion system
Nature, 2009
Type IV secretion systems are secretion nanomachines spanning the two membranes of Gramnegative bacteria. Three proteins, VirB7, VirB9, and VirB10 assemble into a 1.05 MDa core spanning the inner and outer membranes. This core consists of 14 copies of each of the proteins and forms two layers, the I and O layers, inserting in the inner and outer membrane, respectively. Here we present the crystal structure of a ~0.6 MDa outer membrane complex containing the entire O-layer. This structure is the largest determined for an outer membrane channel and is also unprecedented in being composed of three proteins. Unexpectedly, this structure identifies VirB10 as the outer membrane channel with a unique hydrophobic double helical trans-membrane region. This structure establishes VirB10 as the only known protein crossing both membranes of Gramnegative bacteria. Comparison of the cryo-EM and crystallographic structures point to conformational changes regulating channel opening and closing.