Production, crystallization and X-ray diffraction analysis of a complex between a fragment of the TssM T6SS protein and a camelid nanobody (original) (raw)
Related papers
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.
The EMBO Journal
The bacterial Type VI secretion system (T6SS) is a macromolecular machine that injects effectors into prokaryotic and eukaryotic cells. The mode of action of the T6SS is similar to contractile phages: the contraction of a sheath structure pushes a tube topped by a spike into target cells. Effectors are loaded onto the spike or confined into the tube. In enteroaggregative E. coli, the Tle1 phospholipase binds the C-terminal extension of the VgrG trimeric spike. Here we purify the VgrG-Tle1 complex and show that a VgrG trimer binds three Tle1 monomers and inhibits their activity. Using covalent cross-linking coupled to highresolution mass spectrometry we provide information on the sites of contact and further identify the requirement for a Tle1 N-terminal secretion sequence in complex formation. Finally, we report the 2.6-Å resolution cryo-electron microscopy tri-dimensional structure of the (VgrG) 3-(Tle1) 3 complex revealing how the effector binds its cargo, and how VgrG inhibits Tle1 phospholipase activity. The inhibition of Tle1 phospholipase activity once bound to VgrG suggests that Tle1 dissociation from VgrG is required upon delivery.
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.
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
Journal of Molecular Biology, 2016
The Type VI secretion system (T6SS) is a multiprotein complex that delivers toxin effectors 2! in both prokaryotic and eukaryotic cells. It is constituted of a long cytoplasmic structure-the 3! tail-made of stacked Hcp hexamers and wrapped by a contractile sheath. Contraction of the 4! sheath propels the inner tube capped by the VgrG spike protein towards the target cell. This 5! tubular structure is built onto an assembly platform-the baseplate-that is composed of the 6! TssEFGK-VgrG subunits. During the assembly process, the baseplate is recruited to a trans-7! envelope complex comprising the TssJ outer membrane lipoprotein and the TssL and TssM 8! inner membrane proteins. This membrane complex serves as docking station for the 9! baseplate/tail and as channel for the passage of the inner tube during sheath contraction. The 10! baseplate is recruited to the membrane complex through multiple contacts including 11! interactions of TssG and TssK with the cytoplasmic loop of TssM, and TssK interaction with 12! the cytoplasmic domain of TssL, TssL Cyto. Here, we show that TssL Cyto interacts also with the 13! TssE baseplate subunit. Based on the available TssL Cyto structures, we targeted conserved 14! regions and specific features of TssL Cyto in enteroaggregative Escherichia coli (EAEC). By 15! using bacterial two-hybrid and co-immunoprecipitation, we further show that the disordered 16! L3-L4 loop is necessary to interact with TssK, that the L6-L7 loop mediates the interaction 17! with TssE, whereas the TssM cytoplasmic loop binds the conserved groove of TssL Cyto. 18! Finally, competition assays demonstrated that these interactions are physiologically important 19! for EAEC T6SS function. 20!
Molecular microbiology, 2015
The Type VI secretion system (T6SS) is a multi-protein machine that delivers protein effectors in both prokaryotic and eukaryotic cells, allowing interbacterial competition and virulence. The mechanism of action of the T6SS requires the contraction of a sheath-like structure that propels a needle toward target cells, allowing the delivery of protein effectors. Here, we provide evidence that the entero-aggregative Escherichia coli Sci-1 T6SS is required to eliminate competitor bacteria. We further identify Tle1, a toxin effector encoded by this cluster and showed that Tle1 possesses phospholipase A1 and A2 activities required for the inter-bacterial competition. Self-protection of the attacker cell is secured by an outer membrane lipoprotein, Tli1, which binds Tle1 in a 1:1 stoichiometric ratio with nanomolar affinity, and inhibits its phospholipase activity. Tle1 is delivered into the periplasm of the prey cells using the VgrG1 needle spike protein as carrier. Further analyses demon...
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...
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...
Diverse type VI secretion phospholipases are functionally plastic antibacterial effectors
Nature, 2013
Membranes allow the compartmentalization of biochemical processes and are therefore fundamental to life. The conservation of the cellular membrane, combined with its accessibility to secreted proteins, has made it a common target of factors mediating antagonistic interactions between diverse organisms. Here we report the discovery of a diverse superfamily of bacterial phospholipase enzymes. Within this superfamily, we defined enzymes with phospholipase A1 (PLA 1 ) and A2 (PLA 2 ) activity, which are common in host cell-targeting bacterial toxins and the venoms of certain insects and reptiles 1,2 . However, we find that the fundamental role of the superfamily is to mediate antagonistic bacterial interactions as effectors of the type VI secretion system (T6SS) translocation apparatus; accordingly, we name these proteins type VI lipase effectors (Tle). Our analyses indicate that PldA of Pseudomonas aeruginosa, a eukaryotic-like phospholipase D (PLD) 3 , is a member of the Tle superfamily and the founding substrate of the haemolysin co-regulated protein secretion island II T6SS (H2-T6SS). While prior studies have specifically implicated PldA and the H2-T6SS in pathogenesis 3-5 , we uncovered a specific role for the effector and its secretory machinery in intra-and inter-species bacterial interactions. Furthermore we find that this effector achieves its antibacterial activity by degrading phosphatidylethanolamine (PE), the major component of bacterial membranes. The surprising finding that virulence-associated phospholipases can serve as specific antibacterial effectors suggests that interbacterial interactions are a relevant factor driving the ongoing evolution of pathogenesis.