scholarly journals TssA from Aeromonas hydrophila: expression, purification and crystallographic studies

2018 ◽  
Vol 74 (9) ◽  
pp. 578-582 ◽  
Author(s):  
Samuel R. Dix ◽  
Ruyue Sun ◽  
Matthew J. Harris ◽  
Sarah L. Batters ◽  
Svetlana E. Sedelnikova ◽  
...  
Keyword(s):  
Aeromonas Hydrophila ◽  
Secretion System ◽  
Gram Negative Bacteria ◽  
Type Vi Secretion System ◽  
Expression And Purification ◽  
X Ray ◽  
Terminal Domain ◽  
Type Vi Secretion ◽  
Middle Domain ◽  
Major Player

TssA is a core subunit of the type VI secretion system, which is a major player in interspecies competition in Gram-negative bacteria. Previous studies on enteroaggregative Escherichia coli TssA suggested that it is comprised of three putative domains: a conserved N-terminal domain, a middle domain and a ring-forming C-terminal domain. X-ray studies of the latter two domains have identified their respective structures. Here, the results of the expression and purification of full-length and domain constructs of TssA from Aeromonas hydrophila are reported, resulting in diffraction-quality crystals for the middle domain (Nt2) and a construct including the middle and C-terminal domains (Nt2-CTD).

scholarly journals A Type VI Secretion System Effector Protein, VgrG1, from Aeromonas hydrophila That Induces Host Cell Toxicity by ADP Ribosylation of Actin

2009 ◽  
Vol 192 (1) ◽  
pp. 155-168 ◽  
Author(s):  
G. Suarez ◽  
J. C. Sierra ◽  
T. E. Erova ◽  
J. Sha ◽  
A. J. Horneman ◽  
...  
Keyword(s):  
Host Cell ◽  
Aeromonas Hydrophila ◽  
Secretion System ◽  
Full Length ◽  
Spectrometric Analysis ◽  
Type Vi Secretion System ◽  
Terminal Domain ◽  
Type Vi Secretion ◽  
Carboxyl Terminal Domain ◽  
Carboxyl Terminal

ABSTRACT We recently delineated the importance of a type VI secretion system (T6SS) gene cluster in the virulence of diarrheal isolate SSU of Aeromonas hydrophila and showed that VasH, a σ54 activator and T6SS component, was involved in the production of its associated effectors, e.g., hemolysin-coregulated protein. To identify additional T6SS effectors and/or secreted proteins, we subjected culture supernatants from deletion mutants of A. hydrophila, namely, a Δact mutant (a T2SS-associated cytotoxic enterotoxin-encoding gene) and a Δact ΔvasH mutant, to 2-dimensional gel electrophoresis and mass spectrometric analysis. Based on these approaches, we identified a member of the VgrG protein family, VgrG1, that contained a vegetative insecticidal protein (VIP-2) domain at its carboxyl-terminal end. Consequently, the vgrG1 gene was cloned in pBI-EGFP and pET-30a vectors to be expressed in HeLa Tet-Off cells and Escherichia coli, respectively. We assessed the ADP-ribosyltransferase (ADPRT) activity of various domains of purified recombinant VgrG1 (rVgrG1) and provided evidence that only the full-length VgrG1, as well as its carboxyl-terminal domain encoding the VIP-2 domain, showed ADPRT activity. Importantly, bacterium-host cell interaction was needed for the T6SS to induce cytotoxicity in eukaryotic cells, and we demonstrated translocation of VgrG1. Furthermore, our data indicated that expression of the genes encoding the full-length VgrG1 and its carboxyl-terminal domain in HeLa Tet-Off cells disrupted the actin cytoskeleton, which was followed by a decrease in cell viability and an increase in apoptosis. Taken together, these findings demonstrated for the first time that VgrG1 of A. hydrophila possessed actin ADPRT activity associated with its VIP-2 domain and that this domain alone was able to induce a rounded phenotype in HeLa Tet-Off cells, followed by apoptosis mediated by caspase 9 activation.

scholarly journals Diversification of the Type VI Secretion System in Agrobacteria

mBio ◽  
2021 ◽  
Author(s):  
Chih-Feng Wu ◽  
Alexandra J. Weisberg ◽  
Edward W. Davis ◽  
Lin Chou ◽  
Surtaz Khan ◽  
...  
Keyword(s):  
Secretion System ◽  
Effector Proteins ◽  
Gram Negative Bacteria ◽  
Type Vi Secretion System ◽  
Gram Negative ◽  
Bacterial Interactions ◽  
Type Vi Secretion

The T6SS is used by several taxa of Gram-negative bacteria to secrete toxic effector proteins to attack others. Diversification of effector collections shapes bacterial interactions and impacts the health of hosts and ecosystems in which bacteria reside.

scholarly journals Structure of a Peptidoglycan Amidase Effector Targeted to Gram-Negative Bacteria by the Type VI Secretion System

Cell Reports ◽  
2012 ◽  
Vol 1 (6) ◽  
pp. 656-664 ◽  
Author(s):  
Seemay Chou ◽  
Nhat Khai Bui ◽  
Alistair B. Russell ◽  
Katrina W. Lexa ◽  
Taylor E. Gardiner ◽  
...  
Keyword(s):  
Secretion System ◽  
Gram Negative Bacteria ◽  
Type Vi Secretion System ◽  
Gram Negative ◽  
Type Vi Secretion

scholarly journals Cloning, purification, crystallization and preliminary X-ray studies of the putative type VI secretion immunity protein Tli5 (PA5088) fromPseudomonas aeruginosa

2014 ◽  
Vol 70 (7) ◽  
pp. 903-905 ◽  
Author(s):  
Zhen Chen ◽  
Zengqiang Gao ◽  
Haidai Hu ◽  
Jianhua Xu ◽  
Heng Zhang ◽  
...  
Keyword(s):  
Crystallographic Analysis ◽  
Secretion System ◽  
Type Vi Secretion System ◽  
X Ray Diffraction ◽  
Unit Cell Parameters ◽  
Immunity Protein ◽  
X Ray ◽  
Cell Parameters ◽  
Putative Protein ◽  
Type Vi Secretion

The putative protein PA5089 fromPseudomonas aeruginosahas recently been identified as a Tle5 phospholipase effector from a type VI secretion system (T6SS), and its toxicity can be neutralized by the cognate immunity protein Tli5 (PA5088). Here, the expression, purification, crystallization and preliminary crystallographic analysis of PA5088 are reported. X-ray diffraction data were collected from selenomethionine-derivatized PA5088 crystals to a resolution of 2.55 Å. The crystals belonged to space groupP21, with unit-cell parametersa= 64.002,b= 104.744,c= 90.168 Å.

scholarly journals The Type VI Secretion System Spike Protein VgrG5 Mediates Membrane Fusion during Intercellular Spread by Pseudomallei Group Burkholderia Species

2014 ◽  
Vol 82 (4) ◽  
pp. 1436-1444 ◽  
Author(s):  
Isabelle J. Toesca ◽  
Christopher T. French ◽  
Jeff F. Miller
Keyword(s):  
Membrane Fusion ◽  
Plasma Membranes ◽  
Secretion System ◽  
Adjacent Cell ◽  
Type Vi Secretion System ◽  
Content Type ◽  
Terminal Domain ◽  
Fusogenic Activity ◽  
Type Vi Secretion ◽  
Intercellular Spread

ABSTRACTPseudomallei groupBurkholderiaspecies are facultative intracellular parasites that spread efficiently from cell to cell by a mechanism involving the fusion of adjacent cell membranes. Intercellular fusion requires the function of the cluster 5 type VI secretion system (T6SS-5) and its associated valine-glycine repeat protein, VgrG5. Here we show that VgrG5 alleles are conserved and functionally interchangeable betweenBurkholderia pseudomalleiand its relativesB. mallei,B. oklahomensis, andB. thailandensis. We also demonstrate that the integrity of the VgrG5 C-terminal domain is required for fusogenic activity, and we identify sequence motifs, including two hydrophobic segments, that are important for fusion. Mutagenesis and secretion experiments usingB. pseudomalleistrains engineered to express T6SS-5in vitroshow that the VgrG5 C-terminal domain is dispensable for T6SS-mediated secretion of Hcp5, demonstrating that the ability of VgrG5 to mediate membrane fusion can be uncoupled from its essential role in type VI secretion. We propose a model in which a unique fusogenic activity at the C terminus of VgrG5 facilitates intercellular spread byB. pseudomalleiand related species following injection across the plasma membranes of infected cells.

scholarly journals Bacterial Type VI secretion system could have evolved from co-opted tail sheath tube of bacteriophages

2019 ◽  
Author(s):  
Wenfa Ng
Keyword(s):  
Protein Structure ◽  
Bacterial Genome ◽  
Secretion System ◽  
Gram Negative Bacteria ◽  
Bacterial Cells ◽  
Type Vi Secretion System ◽  
Gram Negative ◽  
Type Vi Secretion ◽  
Phage Tail ◽  
Tail Protein

Bacterial cells utilize a variety of nanomachines to secrete proteins and other molecules into the extracellular environment or target cells. One example is the Type VI secretion system (T6SS) in Gram-negative bacteria. Armed with a contractile mechanism similar to that used by bacteriophages to inject phage DNA into bacterial cells, the T6SS shares a common evolutionary origin with tail associated proteins of bacteriophages at both the structural and protein composition levels. Specifically, proteins constituting the T6SS are known to share provenance with those of the phage tail protein. More importantly, the T6SS is strikingly similar to the phage tail protein in both structure and function. However, a more important question concerns whether the T6SS evolved from the phage tail protein and if yes, what is the mechanism responsible for its development? One possibility could be the co-opt of the tail protein structure by bacterial cells through integration of the genes encoding the tail protein structure within the bacterial genome. In this case, expression of the phage tail protein genes would have resulted in a multiprotein structure without apparent function, which meant that a significant gap remains in comparison with extant T6SS that spans the inner and outer cell membrane of Gram-negative bacteria. While it is desirable to trace the evolutionary steps taken by phage tail proteins to transform into functional T6SS, multiple selection pressure and strong mutational propensity might have erased molecular evidence of such transformation. Hence, the challenge lies in uncovering as much structural and sequence evidence as possible that points to distinct steps in the evolutionary pathway towards T6SS. Structural studies offer a particularly promising route to unentangle the details but it must be augmented with sequence evidence that pins down the molecular events that shape the evolution of the complex multiprotein structure, where clefts from one protein fit into the folds of another in yielding a function that could evolve over eons. Collectively, structural and functional similarity between T6SS and phage tail protein suggests a common evolutionary origin for both macromolecular complexes, which has been established through combined structural, compositional and sequence analysis. But the steps underpinning the transformation of phage tail protein into T6SS remain unclear, which obfuscate understanding of the evolutionary forces that shape the transformation. One possible evolutionary trajectory posits that genes expressing phage tail proteins were co-opted and integrated into the bacterial genome. However, significant gap remains between a phage tail protein structure with unclear function in the cytoplasm and a functional T6SS that spans two bacterial membranes. Future detective work at the structural and sequence level might offer clues to the evolutionary path trodden by a precursor of the bacterial T6SS.

scholarly journals Structure of a bacterial Rhs effector exported by the type VI secretion system

2021 ◽  
Author(s):  
Patrick Guenther ◽  
Dennis Quentin ◽  
Shehryar Ahmad ◽  
Kartik Sachar ◽  
Christos Gatsogiannis ◽  
...  
Keyword(s):  
Bacterial Cell ◽  
Secretion System ◽  
Effector Proteins ◽  
Protein Export ◽  
Spike Protein ◽  
Gram Negative Bacteria ◽  
Structural Elements ◽  
Type Vi Secretion System ◽  
Gram Negative ◽  
Type Vi Secretion

The type VI secretion system (T6SS) is a widespread protein export apparatus found in Gram-negative bacteria. The majority of T6SSs deliver toxic effector proteins into competitor bacteria. Yet, the structure, function, and activation of many of these effectors remains poorly understood. Here, we present the structures of the T6SS effector RhsA from Pseudomonas protegens and its cognate T6SS spike protein, VgrG1, at 3.3 Å resolution. The structures reveal that the rearrangement hotspot (Rhs) repeats of RhsA assemble into a closed anticlockwise β-barrel spiral similar to that found in bacterial insecticidal Tc toxins and in metazoan teneurin proteins. We find that the C-terminal toxin domain of RhsA is autoproteolytically cleaved but remains inside the Rhs ′cocoon′ where, with the exception of three ordered structural elements, most of the toxin is disordered. The N-terminal ′plug′ domain is unique to T6SS Rhs proteins and resembles a champagne cork that seals the Rhs cocoon at one end while also mediating interactions with VgrG1. Interestingly, this domain is also autoproteolytically cleaved inside the cocoon but remains associated with it. We propose that mechanical force is required to remove the cleaved part of the plug, resulting in the release of the toxin domain as it is delivered into a susceptible bacterial cell by the T6SS.

scholarly journals Killing of Gram-negative and Gram-positive bacteria by a bifunctional cell wall-targeting T6SS effector

2021 ◽  
Vol 118 (40) ◽  
pp. e2106555118
Author(s):  
Nguyen-Hung Le ◽  
Victor Pinedo ◽  
Juvenal Lopez ◽  
Felipe Cava ◽  
Mario F. Feldman
Keyword(s):  
Secretion System ◽  
Bifunctional Enzyme ◽  
Gram Negative Bacteria ◽  
Type Vi Secretion System ◽  
Gram Positive ◽  
Gram Negative ◽  
Bacterial Interactions ◽  
Gram Positive Bacteria ◽  
Lytic Transglycosylase ◽  
Type Vi Secretion

The type VI secretion system (T6SS) is a powerful tool deployed by Gram-negative bacteria to antagonize neighboring organisms. Here, we report that Acinetobacter baumannii ATCC 17978 (Ab17978) secretes D-lysine (D-Lys), increasing the extracellular pH and enhancing the peptidoglycanase activity of the T6SS effector Tse4. This synergistic effect of D-Lys on Tse4 activity enables Ab17978 to outcompete Gram-negative bacterial competitors, demonstrating that bacteria can modify their microenvironment to increase their fitness during bacterial warfare. Remarkably, this lethal combination also results in T6SS-mediated killing of Gram-positive bacteria. Further characterization revealed that Tse4 is a bifunctional enzyme consisting of both lytic transglycosylase and endopeptidase activities, thus representing a family of modularly organized T6SS peptidoglycan-degrading effectors with an unprecedented impact in antagonistic bacterial interactions.

scholarly journals Roles of Type VI Secretion System in Transport of Metal Ions

2021 ◽  
Vol 12 ◽  
Author(s):  
Xiaobing Yang ◽  
Hai Liu ◽  
Yanxiong Zhang ◽  
Xihui Shen
Keyword(s):  
Ion Transport ◽  
Metal Ion ◽  
Transmembrane Protein ◽  
Secretion System ◽  
Gram Negative Bacteria ◽  
Type Vi Secretion System ◽  
Host Interactions ◽  
Extracellular Milieu ◽  
Type Vi Secretion ◽  
Metal Ion Transport

The type VI secretion system (T6SS) is a transmembrane protein nanomachine employed by many gram-negative bacteria to directly translocate effectors into adjacent cells or the extracellular milieu, showing multiple functions in both interbacterial competition and bacteria-host interactions. Metal ion transport is a newly discovered T6SS function. This review summarizes the identified T6SS functions and highlights the features of metal ion transport mediated by T6SS and discusses its regulation.

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