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.

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

EMBO Reports ◽  
2017 ◽  
Vol 18 (7) ◽  
pp. 1090-1099 ◽  
Author(s):  
Yi‐Wei Chang ◽  
Lee A Rettberg ◽  
Davi R Ortega ◽  
Grant J Jensen
Keyword(s):  
Secretion System ◽  
Type Vi Secretion System ◽  
Type Vi Secretion ◽  
Electron Cryotomography

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 In Situ Visualization of the pKM101-Encoded Type IV Secretion System Reveals a Highly Symmetric ATPase Energy Center at the Channel Entrance

2021 ◽  
Author(s):  
Pratick Khara ◽  
Peter J. Christie ◽  
Bo Hu
Keyword(s):  
Cell Envelope ◽  
Secretion System ◽  
Type Iv Secretion ◽  
Type Iv Secretion System ◽  
Membrane Complex ◽  
Type Iv ◽  
Inner Membrane Complex ◽  
Cytoplasmic Complex

Bacterial conjugation systems are members of the type IV secretion system (T4SS) superfamily. T4SSs can be classified as ‘minimized’ or ‘expanded’ based on whether assembly requires only a core set of signature subunits or additional system-specific components. The prototypical ‘minimized’ systems mediating Agrobacterium tumefaciens T-DNA transfer and conjugative transfer of plasmids pKM101 and R388 are built from 12 subunits generically named VirB1-VirB11 and VirD4. In this study, we visualized the pKM101-encoded T4SS in the native context of the bacterial cell envelope by in situ cryoelectron tomography (CryoET). The T4SSpKM101 is composed of an outer membrane core complex (OMCC) connected by a thin stalk to an inner membrane complex (IMC). The OMCCexhibits 14-fold symmetry and resembles that of the T4SSR388, a large substructure of which was previously purified and analyzed by negative-stain electron microscopy (nsEM). The IMC of the in situ T4SSpKM101 machine is highly symmetrical and exhibits 6-fold symmetry, dominated by a hexameric collar in the periplasm and a cytoplasmic complex composed of a hexamer of dimers of the VirB4-like TraB ATPase. The IMCclosely resembles equivalent regions of three ‘expanded’ T4SSs previously visualized by in situ CryoET, but strikingly differs from the IMC of the purified T4SSR388 whose cytoplasmic complex instead presents as two side-by-side VirB4 hexamers.  Together, our findings support a unified architectural model for all T4SSs assembled in vivo regardless of their classification as ‘minimized’ or ‘expanded’: the signature VirB4-like ATPases invariably are arranged as central hexamers of dimers at the entrances to the T4SS channels.

scholarly journals Quantitative description of a contractile macromolecular machine

2021 ◽  
Vol 7 (24) ◽  
pp. eabf9601
Author(s):  
Alec Fraser ◽  
Nikolai S. Prokhorov ◽  
Fang Jiao ◽  
B. Montgomery Pettitt ◽  
Simon Scheuring ◽  
...  
Keyword(s):  
Lipid Membranes ◽  
Cell Walls ◽  
Structural Information ◽  
Quantitative Description ◽  
Secretion System ◽  
Combined Approach ◽  
Type Vi Secretion System ◽  
Penetration Process ◽  
Rigid Tube ◽  
Type Vi Secretion

Contractile injection systems (CISs) [type VI secretion system (T6SS), phage tails, and tailocins] use a contractile sheath-rigid tube machinery to breach cell walls and lipid membranes. The structures of the pre- and postcontraction states of several CISs are known, but the mechanism of contraction remains poorly understood. Combining structural information of the end states of the 12-megadalton R-type pyocin sheath-tube complex with thermodynamic and force spectroscopy analyses and an original modeling procedure, we describe the mechanism of pyocin contraction. We show that this nanomachine has an activation energy of 160 kilocalories/mole (kcal/mol), and it releases 2160 kcal/mol of heat and develops a force greater than 500 piconewtons. Our combined approach provides a quantitative and experimental description of the membrane penetration process by a CIS.

scholarly journals The double tubular contractile structure of the type VI secretion system displays striking flexibility and elasticity

2018 ◽  
Author(s):  
Maria Silvina Stietz ◽  
Xiaoye Liang ◽  
Megan Wong ◽  
Steven Hersch ◽  
Tao G. Dong
Keyword(s):  
Cell Envelope ◽  
Intermediate Stage ◽  
Secretion System ◽  
Physical Contact ◽  
Bacterial Cells ◽  
Type Vi Secretion System ◽  
Type Vi Secretion ◽  
Outer Sheath ◽  
Sufficient Power ◽  
Mechanical Pressing

AbstractThe double tubular structure of the type VI secretion system (T6SS) is considered as one of the longest straight and rigid intracellular structures in bacterial cells. Contraction of the T6SS outer sheath occurs almost instantly and releases sufficient power to inject the inner needle-like Hcp tube and its associated effectors into target bacterial cells through piercing the stiff cell envelope. The molecular mechanism triggering T6SS contraction remains elusive. Here we report that the double tubular T6SS structure is strikingly flexible and elastic, forming U-, circular-, or S-shapes while maintaining functional for contraction and substrate delivery. We show that physical contact with cytoplasmic membrane induced a range of T6SS structure deformation, but the resultant mechanical pressing force on the T6SS baseplate did not trigger contraction. Our results also reveal a stalling intermediate stage of sheath-tube extension following which the structure contracts or resumes to extend. These observations suggest that the recruitment equilibrium of sheath-tube precursors to the extending structure is key to stability/contraction and lead us to propose a model of T6SS contraction, termed ESCAPE (extension-stall-contraction and precursor equilibrium). Our data highlight the remarkable flexibility of the double tubular T6SS structure and its length control mechanism distinct from the other evolutionarily related contractile cell-puncturing systems.

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 Structural insights into the function of type VI secretion system TssA subunits

2018 ◽  
Vol 9 (1) ◽  
Author(s):  
Samuel R. Dix ◽  
Hayley J. Owen ◽  
Ruyue Sun ◽  
Asma Ahmad ◽  
Sravanthi Shastri ◽  
...  
Keyword(s):  
Secretion System ◽  
Type Vi Secretion System ◽  
Type Vi Secretion ◽  
Structural Insights

Citrobacter freundii Activation of NLRP3 Inflammasome via the Type VI Secretion System

2020 ◽  
Author(s):  
Liyun Liu ◽  
Liqiong Song ◽  
Rong Deng ◽  
Ruiting Lan ◽  
Wenjie Jin ◽  
...  
Keyword(s):  
Nlrp3 Inflammasome ◽  
Secretion System ◽  
Interleukin 1Β ◽  
Inflammasome Activation ◽  
Dependent Manner ◽  
Citrobacter Freundii ◽  
Type Vi Secretion System ◽  
Type Vi Secretion ◽  
Tract Infections

Abstract Citrobacter freundii is a significant cause of human infections, responsible for food poisoning, diarrhea, and urinary tract infections. We previously identified a highly cytotoxic and adhesive C. freundii strain CF74 expressing a type VI secretion system (T6SS). In this study, we showed that in mice-derived macrophages, C. freundii CF74 activated the Nucleotide Oligomerization Domain -Like Receptor Family, Pyrin Domain Containing 3(NLRP3) inflammasomes in a T6SS-dependent manner. The C. freundii T6SS activated the inflammasomes mainly through caspase 1 and mediated pyroptosis of macrophages by releasing the cleaved gasdermin-N domain. The CF74 T6SS was required for flagellin-induced interleukin 1β release by macrophages. We further show that the T6SS tail component and effector, hemolysin co-regulation protein-2 (Hcp-2), was necessary and sufficient to trigger NLRP3 inflammasome activation. In vivo, the T6SS played a key role in mediating interleukin 1β secretion and the survival of mice during C. freundii infection in mice. These findings provide novel insights into the role of T6SS in the pathogenesis of C. freundii.

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