Role and recruitment of the TagL peptidoglycan-binding protein during Type VI secretion system biogenesis

International audienceThe type VI secretion system (T6SS) is an injection apparatus that uses a springlike mechanism for effector delivery. The contractile tail is composed of a needle tipped by a sharpened spike and wrapped by the sheath that polymerizes in an extended conformation on the assembly platform, or baseplate. Contraction of the sheath propels the needle and effectors associated with it into target cells. The passage of the needle through the cell envelope of the attacker is ensured by a dedicated trans-envelope channel complex. This membrane complex (MC) comprises the TssJ lipoprotein and the TssL and TssM inner membrane proteins. MC assembly is a hierarchized mechanism in which the different subunits are recruited in a specific order: TssJ, TssM, and then TssL. Once assembled, the MC serves as a docking station for the baseplate. In enteroaggregative Escherichia coli, the MC is accessorized by TagL, a peptidoglycan-binding (PGB) inner membrane-anchored protein. Here, we show that the PGB domain is the only functional domain of TagL and that the N-terminal transmembrane region mediates contact with the TssL transmembrane helix. Finally, we conduct fluorescence microscopy experiments to position TagL in the T6SS biogenesis pathway, demonstrating that TagL is recruited to the membrane complex downstream of TssL and is not required for baseplate docking

Tryptophan-mediated Dimerization of the TssL Transmembrane Anchor Is Required for Type VI Secretion System Activity

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Chapter 23 Defining assembly pathways by fluorescence microscopy

International audienceBacterial secretion systems are amongst the largest protein complexes in prokaryotes, and display remarkably complex architectures. Their assembly often follows clearly defined pathways. Deciphering these pathways not only reveals how bacteria accomplish to build these large functional complexes, but can provide crucial information on the interactions and subcomplexes within secretion systems, their distribution within the bacterium, and even functional insights. The emergence of fluorescent proteins has provided a new powerful tool for biological imaging, and the use of fluorescently labeled components presents an interesting method to accurately define the biogenesis of macromolecular complexes. Here, we describe the use of this method to decipher the assembly pathway of bacterial secretion systems

Biogenesis and membrane anchoring of the Type VI secretion contractile tail

Récemment, le système de sécrétion de type VI (SST6) a été identifié comme un nouvel acteur clé dans la compétition inter-bactérienne parmi le large arsenal dont dispose les bactéries. L’une des particularités du SST6 est de cibler à la fois des cellules eucaryotes et procaryotes. Le T6SS est un complexe protéique formé par l’assemblage de deux ‘sous-complexes’. Le premier sert à l’ancrage de la machinerie au sein de l’enveloppe bactérienne et le second agit comme une arbalète moléculaire. Le mécanisme d’action du SST6 est très similaire à celui d’autres machineries contractiles telles que celui des bactériophages : la contraction d’un fourreau propulse une flèche, composée d’un tube avec une aiguille à son extrémité, directement dans la cellule cible afin de délivrer les différentes toxines. Mon projet de thèse consiste à comprendre quelles sont la structure et la biogénèse des deux différents complexes et de comprendre comment ils sont assemblés. Nous utilisons comme modèle la bactérie pathogène à Gram négatif Escherichia coli entéroagrégative. J’ai pu démontrer que le complexe membranaire est assemblé en premier, avec l’adressage de la lipoprotéine de membrane externe TssJ, puis le recrutement séquentiel de TssM et TssL, deux protéines de membrane interne. Le complexe membranaire recrute ensuite une plateforme d’assemblage, appelée ‘baseplate’. Nous avons identifié et caractérisé les composants de cette ‘baseplate’ qui sert de plateforme d’assemblage pour le recrutement du reste de la machinerie (fourreau et flèche). Enfin, nous avons identifié et déterminé le rôle de la protéine TssA, une protéine qui coordonne la polymérisation du fourreau et de la flèche.Among the broad weaponry of bacteria, the recently identified type VI secretion system (T6SS) emerges as one of the key player in bacterial competition. T6SS is a versatile machinery that targets both eukaryotic and prokaryotic cells. This molecular weapon assembles two evolutionarily different sub-assemblies. One complex anchors the machinery to the cell envelope while the second acts as a molecular crossbow. The mechanism of action of the T6SS is similar to other known contractile machineries such as bacteriophages: the contraction of a sheath propels an arrow, constituted of a tail tube capped by a cell-puncturing device, directly into the prey cell to deliver effector toxins. My Ph.D project was to provide mechanistic details on the structure and biogenesis of the two T6SS sub-complexes and to understand how they are connected, using entero-aggregative Escherichia coli as model bacterium. I have demonstrated that the membrane complex is assembled first and starts with the positioning of the outer membrane TssJ lipoprotein and proceeds inward, from the outer to the inner membrane, through the sequential recruitment of the TssM and TssL subunits. After assembly, the membrane complex recruits an assembly platform called the baseplate. We identified and characterized the components of this baseplate, which serves as assembly platform for the tail. We further demonstrated that the functional and physical interaction between the T6SS membrane complex and the baseplate is mediated by multiple contacts. Finally, we identified and deciphered the role of TssA, a protein that coordinates the polymerizations of the tail tube and sheath

Chapter 16. Fusion reporter approaches to monitor transmembrane helix interactions in bacterial membranes Running head: protein-protein interaction in the membrane

International audienceIn transenvelope multiprotein machines such as bacterial secretion systems, protein-protein interactions not only occur between soluble domains but are also mediated by helix-helix contacts in the inner membrane. Here, we describe genetic assays commonly used to test interactions between transmembrane α-helices in their native membrane environment. These assays are based on the reconstitution of dimeric regulators allowing the expression of reporter genes. We provide detailed protocols for the TOXCAT and GALLEX assays used to monitor homotypic and heterotypic transmembrane helix-helix interactions

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

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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

International audienceThe 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

The C‐tail anchored TssL subunit, an essential protein of the enteroaggregative Escherichia coli Sci‐1 Type VI secretion system, is inserted by YidC

International audienceType VI secretion systems (T6SS) are macromolecular complexes present in Gram-negative bacteria. T6SS are structurally similar to the bacteriophage cell-puncturing device and have been shown to mediate bacteria–host or bacteria–bacteria interactions. T6SS assemble from 13 to 20 proteins. In enteroaggregative Escherichia coli (EAEC), one of the subassemblies is composed of four proteins that form a trans-envelope complex: the TssJ outer membrane lipoprotein, the peptidoglycan-anchored inner membrane TagL protein, and two putative inner membrane proteins, TssL and TssM. In this study, we characterized the TssL protein of the EAEC Sci-1 T6SS in terms of localization, topology, and function. TssL is a critical component of the T6SS, anchored to the inner membrane through a single transmembrane segment located at the extreme C-terminus of the protein. We further show that this transmembrane segment is essential for the function of the protein and its proper insertion in the inner membrane is dependent upon YidC and modulated by the Hsp70 homologue DnaK

Structure–Function Analysis of the TssL Cytoplasmic Domain Reveals a New Interaction between the Type VI Secretion Baseplate and Membrane Complexes

International audienceThe Type VI secretion system (T6SS) is a multiprotein complex that delivers toxin effectors in both prokaryotic and eukaryotic cells. It is constituted of a long cytoplasmic structure - the tail - made of stacked Hcp hexamers and wrapped by a contractile sheath. Contraction of the sheath propels the inner tube capped by the VgrG spike protein towards the target cell. This tubular structure is built onto an assembly platform - the baseplate - that is composed of the TssEFGK-VgrG subunits. During the assembly process, the baseplate is recruited to a trans-envelope complex comprising the TssJ outer membrane lipoprotein and the TssL and TssM inner membrane proteins. This membrane complex serves as docking station for the baseplate/tail and as channel for the passage of the inner tube during sheath contraction. The baseplate is recruited to the membrane complex through multiple contacts including interactions of TssG and TssK with the cytoplasmic loop of TssM, and TssK interaction with the cytoplasmic domain of TssL, TssLCyto. Here, we show that TssLCyto interacts also with the TssE baseplate subunit. Based on the available TssLCyto structures, we targeted conserved regions and specific features of TssLCyto in enteroaggregative Escherichia coli (EAEC). By using bacterial two-hybrid and co-immunoprecipitation, we further show that the disordered L3-L4 loop is necessary to interact with TssK, that the L6-L7 loop mediates the interaction with TssE, whereas the TssM cytoplasmic loop binds the conserved groove of TssLCyto. Finally, competition assays demonstrated that these interactions are physiologically important for EAEC T6SS function

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

International audienceThe 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