scholarly journals Anchoring the T6SS to the cell wall: Crystal structure of the peptidoglycan binding domain of the TagL accessory protein

PLoS ONE ◽  
2021 ◽  
Vol 16 (7) ◽  
pp. e0254232
Author(s):  
Van Son Nguyen ◽  
Silvia Spinelli ◽  
Éric Cascales ◽  
Alain Roussel ◽  
Christian Cambillau ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Cell Wall ◽  
Protein Delivery ◽  
Cell Envelope ◽  
Interaction Network ◽  
Binding Domain ◽  
Target Cells ◽  
Accessory Protein ◽  
Type Vi Secretion System ◽  
Membrane Complex

The type VI secretion system (T6SS) is a widespread mechanism of protein delivery into target cells, present in more than a quarter of all sequenced Gram-negative bacteria. The T6SS constitutes an important virulence factor, as it is responsible for targeting effectors in both prokaryotic and eukaryotic cells. The T6SS comprises a tail structure tethered to the cell envelope via a trans-envelope complex. In most T6SS, the membrane complex is anchored to the cell wall by the TagL accessory protein. In this study, we report the first crystal structure of a peptidoglycan-binding domain of TagL. The fold is conserved with members of the OmpA/Pal/MotB family, and more importantly, the peptidoglycan binding site is conserved. This structure further exemplifies how proteins involved in anchoring to the cell wall for different cellular functions rely on an interaction network with peptidoglycan strictly conserved.

scholarly journals Assembly and Subcellular Localization of Bacterial Type VI Secretion Systems

2019 ◽  
Vol 73 (1) ◽  
pp. 621-638 ◽  
Author(s):  
Jing Wang ◽  
Maj Brodmann ◽  
Marek Basler
Keyword(s):  
Subcellular Localization ◽  
Cell Envelope ◽  
Target Cells ◽  
Type Vi Secretion System ◽  
Secretion Systems ◽  
Large Molecules ◽  
Membrane Complex ◽  
Type Vi Secretion ◽  
A Cell ◽  
Protein Secretion Systems

Bacteria need to deliver large molecules out of the cytosol to the extracellular space or even across membranes of neighboring cells to influence their environment, prevent predation, defeat competitors, or communicate. A variety of protein-secretion systems have evolved to make this process highly regulated and efficient. The type VI secretion system (T6SS) is one of the largest dynamic assemblies in gram-negative bacteria and allows for delivery of toxins into both bacterial and eukaryotic cells. The recent progress in structural biology and live-cell imaging shows the T6SS as a long contractile sheath assembled around a rigid tube with associated toxins anchored to a cell envelope by a baseplate and membrane complex. Rapid sheath contraction releases a large amount of energy used to push the tube and toxins through the membranes of neighboring target cells. Because reach of the T6SS is limited, some bacteria dynamically regulate its subcellular localization to precisely aim at their targets and thus increase efficiency of toxin translocation.

scholarly journals Role and Recruitment of the TagL Peptidoglycan-Binding Protein during Type VI Secretion System Biogenesis

2019 ◽  
Vol 201 (12) ◽  
Author(s):  
Yoann G. Santin ◽  
Claire E. Camy ◽  
Abdelrahim Zoued ◽  
Thierry Doan ◽  
Marie-Stéphanie Aschtgen ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cell Envelope ◽  
Secretion System ◽  
Target Cells ◽  
Functional Domain ◽  
Type Vi Secretion System ◽  
Inner Membrane ◽  
Content Type ◽  
Membrane Complex ◽  
Type Vi Secretion

ABSTRACT The 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. IMPORTANCE Bacteria use weapons to deliver effectors into target cells. One of these weapons, called the type VI secretion system (T6SS), could be compared to a nano-spear gun using a springlike mechanism for effector injection. By targeting bacteria and eukaryotic cells, the T6SS reshapes bacterial communities and hijacks host cell defenses. In enteroaggregative Escherichia coli, the T6SS is a multiprotein machine that comprises a cytoplasmic tail and a peptidoglycan-anchored trans-envelope channel. In this work, we show that TagL comprises an N-terminal domain that mediates contact with the channel and a peptidoglycan-binding domain that binds the cell wall. We then determine at which stage of T6SS biogenesis TagL is recruited and how TagL absence impacts the assembly pathway.

scholarly journals Characterization of a lysozyme-like effector TseP and secretion co-dependence of the type VI secretion system in Aeromonas dhakensis SSU

2021 ◽  
Author(s):  
Xiaoye Liang ◽  
Tong-Tong Pei ◽  
Zeng-Hang Wang ◽  
Weiliang Xiong ◽  
Li-Li Wu ◽  
...  
Keyword(s):  
Cell Wall ◽  
Protein Delivery ◽  
Systemic Infection ◽  
Secretion System ◽  
Target Cells ◽  
Type Vi Secretion System ◽  
Secretion Systems ◽  
Bacterial Killing ◽  
Promising Tool ◽  
Type Vi Secretion

The type VI secretion system (T6SS) is a widespread weapon employed by gram-negative bacteria for interspecies interaction in complex communities. Analogous to a contractile phage tail, the double-tubular T6SS injects toxic effectors into prokaryotic and eukaryotic neighboring cells. Although effectors dictate T6SS functions, their identities remain elusive in many pathogens. Here we report a lysozyme-like effector TseP in Aeromonas dhakensis, a waterborne pathogen that can cause severe gastroenteritis and systemic infection. Using secretion, competition and enzymatic assays, we demonstrate that TseP is a T6SS-dependent effector with cell wall-lysing activities and TsiP is its cognate immunity protein. Triple deletion of tseP and two known effector genes tseI and tseC abolished T6SS-mediated secretion, while complementation with any single effector gene partially restores bacterial killing and Hcp secretion. By contrast to whole-gene deletions, a triple-effector-inactivated mutant 3effc showed abolished antibacterial killing but retained T6SS secretion. We further demonstrate that the 3effc mutation abolished T6SS-mediated toxicity of SSU to Dictyostelium discoideum amoeba, suggesting that the T6SS physical puncture is nontoxic to eukaryotic cells. These data highlight not only the necessity of possessing functionally diverse effectors for survival in multispecies communities but also that effector inactivation would be an efficient strategy to detoxify the T6SS while preserving its delivery efficiency, converting the T6SS to a protein delivery platform to a variety of recipient cells. Importance Delivery of cargo proteins via protein secretion systems has been shown as a promising tool in various applications. However, secretion systems are often used by pathogens to cause disease. Thus, strategies are needed to detoxify secretion systems while preserving their efficiency. The T6SS can translocate proteins through physical puncture of target cells without specific surface receptorsand can target a broad range of recipients. In this study, we identified a cell-wall lysing effector and by inactivating it and the other two known effectors, we have built a detoxified T6SS-active strain that may be used for protein delivery towards prokaryotic and eukaryotic recipient cells.

scholarly journals Binding From both sides: TolR and full-length OmpA bind and maintain the local structure of the E. coli cell wall

2018 ◽  
Author(s):  
Alister T. Boags ◽  
Firdaus Samsudin ◽  
Syma Khalid
Keyword(s):  
Cell Wall ◽  
Electrostatic Interactions ◽  
Cell Envelope ◽  
Binding Domain ◽  
Gram Negative Bacteria ◽  
Inner Membrane ◽  
E Coli ◽  
Non Covalent Interactions ◽  
Covalent Interactions

SUMMARYWe present a molecular modeling and simulation study of the of the E. coli cell envelope, with a particular focus on the role of TolR, a native protein of the E. coli inner membrane in interactions with the cell wall. TolR has been proposed to bind to peptidoglycan, but the only structure of this protein thus far is in a conformation in which the putative peptidoglycan binding domain is not accessible. We show that a model of the extended conformation of the protein in which this domain is exposed, binds peptidoglycan largely through electrostatic interactions. We show that non-covalent interactions of TolR and OmpA with the cell wall, from the inner membrane and outer membrane sides respectively, maintain the position of the cell wall even in the absence of Braun’s lipoprotein. When OmpA is truncated to remove the peptidoglycan binding domain, TolR is able to pull the cell wall down towards the inner membrane. The charged residues that mediate the cell-wall interactions of TolR in our simulations, are conserved across a number of species of Gram-negative bacteria.

Crystal structures of UDP-N-acetylmuramic acid L-alanine ligase (MurC) from Mycobacterium bovis with and without UDP-N-acetylglucosamine

2021 ◽  
Vol 77 (5) ◽  
pp. 618-627
Author(s):  
Pil-Won Seo ◽  
Suk-Youl Park ◽  
Andreas Hofmann ◽  
Jeong-Sun Kim
Keyword(s):  
Crystal Structure ◽  
Cell Wall ◽  
Catalytic Activity ◽  
Amino Acid ◽  
Mycobacterium Bovis ◽  
Domain Architecture ◽  
Binding Domain ◽  
Cross Linking ◽  
Nickel Ion ◽  
Binding Loop

Peptidoglycan comprises repeating units of N-acetylmuramic acid, N-acetylglucosamine and short cross-linking peptides. After the conversion of UDP-N-acetylglucosamine (UNAG) to UDP-N-acetylmuramic acid (UNAM) by the MurA and MurB enzymes, an amino acid is added to UNAM by UDP-N-acetylmuramic acid L-alanine ligase (MurC). As peptidoglycan is an essential component of the bacterial cell wall, the enzymes involved in its biosynthesis represent promising targets for the development of novel antibacterial drugs. Here, the crystal structure of Mycobacterium bovis MurC (MbMurC) is reported, which exhibits a three-domain architecture for the binding of UNAM, ATP and an amino acid as substrates, with a nickel ion at the domain interface. The ATP-binding loop adopts a conformation that is not seen in other MurCs. In the UNAG-bound structure of MbMurC, the substrate mimic interacts with the UDP-binding domain of MbMurC, which does not invoke rearrangement of the three domains. Interestingly, the glycine-rich loop of the UDP-binding domain of MbMurC interacts through hydrogen bonds with the glucose moiety of the ligand, but not with the pyrophosphate moiety. These findings suggest that UNAG analogs might serve as potential candidates for neutralizing the catalytic activity of bacterial MurC.

scholarly journals Type VI secretion system: secretion by a contractile nanomachine

2015 ◽  
Vol 370 (1679) ◽  
pp. 20150021 ◽  
Author(s):  
Marek Basler
Keyword(s):  
Live Cell Imaging ◽  
Effector Cell ◽  
Cell Imaging ◽  
Secretion System ◽  
Target Cells ◽  
Gram Negative Bacteria ◽  
Type Vi Secretion System ◽  
Secretion Systems ◽  
Membrane Complex ◽  
Type Vi Secretion

The type VI secretion systems (T6SS) are present in about a quarter of all Gram-negative bacteria. Several key components of T6SS are evolutionarily related to components of contractile nanomachines such as phages and R-type pyocins. The T6SS assembly is initiated by formation of a membrane complex that binds a phage-like baseplate with a sharp spike, and this is followed by polymerization of a long rigid inner tube and an outer contractile sheath. Effectors are preloaded onto the spike or into the tube during the assembly by various mechanisms. Contraction of the sheath releases an unprecedented amount of energy, which is used to thrust the spike and tube with the associated effectors out of the effector cell and across membranes of both bacterial and eukaryotic target cells. Subunits of the contracted sheath are recycled by T6SS-specific unfoldase to allow for a new round of assembly. Live-cell imaging has shown that the assembly is highly dynamic and its subcellular localization is in certain bacteria regulated with a remarkable precision. Through the action of effectors, T6SS has mainly been shown to contribute to pathogenicity and competition between bacteria. This review summarizes the knowledge that has contributed to our current understanding of T6SS mode of action.

scholarly journals In vivo structures of an intact type VI secretion system revealed by electron cryotomography

2017 ◽  
Author(s):  
Yi-Wei Chang ◽  
Lee A. Rettberg ◽  
Grant J. Jensen
Keyword(s):  
Structural Information ◽  
Cell Envelope ◽  
Secretion System ◽  
Tail Fiber ◽  
Type Vi Secretion System ◽  
Membrane Complex ◽  
Type Vi Secretion ◽  
Electron Cryotomography ◽  
Structural Insights

SUMMARYThe type VI secretion system (T6SS) is a versatile molecular weapon used by many bacteria against eukaryotic hosts or prokaryotic competitors. It consists of a cytoplasmic bacteriophage tail-like structure anchored in the bacterial cell envelope via a cytoplasmic baseplate and a periplasmic membrane complex. Rapid contraction of the sheath in the bacteriophage tail-like structure propels an inner tube/spike complex through the target cell envelope to deliver effectors. While structures of purified contracted sheath and purified membrane complex have been solved, because sheaths contract upon cell lysis and purification, no structure is available for the extended sheath. Structural information about the baseplate is also lacking. Here we use electron cryotomography to directly visualize intact T6SS structures inside Myxococcus xanthus cells. Using sub-tomogram averaging, we resolve the structure of the extended sheath and membrane-associated components including the baseplate. Moreover, we identify novel extracellular bacteriophage tail fiber-like antennae. These results provide new structural insights into how the extended sheath prevents premature disassembly and how this sophisticated machine may recognize targets.

In vitro assembly of 2-D crystals from partially purified EA1 protein secreted by Bacillus anthracis strain Delta Sterne-1

1994 ◽  
Vol 52 ◽  
pp. 276-277
Author(s):  
Mary Beth Downs ◽  
Wilson Ribot ◽  
Joseph W. Farchaus
Keyword(s):  
Cell Wall ◽  
Molecular Sieves ◽  
Cell Envelope ◽  
Single Species ◽  
Supporting Cell ◽  
Surface Layers ◽  
Rabbit Igg ◽  
In Vitro Assembly ◽  
Covalently Linked

Many bacteria possess surface layers (S-layers) that consist of a two-dimensional protein lattice external to the cell envelope. These S-layer arrays are usually composed of a single species of protein or glycoprotein and are not covalently linked to the underlying cell wall. When removed from the cell, S-layer proteins often reassemble into a lattice identical to that found on the cell, even without supporting cell wall fragments. S-layers exist at the interface between the cell and its environment and probably serve as molecular sieves that exclude destructive macromolecules while allowing passage of small nutrients and secreted proteins. Some S-layers are refractory to ingestion by macrophages and, generally, bacteria are more virulent when S-layers are present.When grown in rich medium under aerobic conditions, B. anthracis strain Delta Sterne-1 secretes large amounts of a proteinaceous extractable antigen 1 (EA1) into the growth medium. Immunocytochemistry with rabbit polyclonal anti-EAl antibody made against the secreted protein and gold-conjugated goat anti-rabbit IgG showed that EAI was localized at the cell surface (fig 1), which suggests its role as an S-layer protein.

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