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 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 Roles of Cyclic AMP Receptor Protein and the Carboxyl-Terminal Domain of the α Subunit in Transcription Activation of theEscherichia coli rhaBAD Operon

2000 ◽  
Vol 182 (12) ◽  
pp. 3529-3535 ◽  
Author(s):  
Carolyn C. Holcroft ◽  
Susan M. Egan
Keyword(s):  
Dna Binding ◽  
Cyclic Amp ◽  
Full Length ◽  
Receptor Protein ◽  
Α Subunit ◽  
Cyclic Amp Receptor Protein ◽  
Terminal Domain ◽  
Carboxyl Terminal Domain ◽  
Plasmid Library ◽  
Carboxyl Terminal

ABSTRACT The Escherichia coli rhaBAD operon encodes the enzymes for catabolism of the sugar l-rhamnose. FullrhaBAD activation requires the AraC family activator RhaS (bound to a site that overlaps the −35 region of the promoter) and the cyclic AMP receptor protein (CRP; bound immediately upstream of RhaS at −92.5). We tested alanine substitutions in activating regions (AR) 1 and 2 of CRP for their effect onrhaBAD activation. Some, but not all, of the substitutions in both AR1 and AR2 resulted in approximately twofold defects in expression from rhaBAD promoter fusions. We also expressed a derivative of the α subunit of RNA polymerase deleted for the entire C-terminal domain (α-Δ235) and assayed expression from rhaBAD promoter fusions. The greatest defect (54-fold) occurred at a truncated promoter where RhaS was the only activator, while the defect at the full-length promoter (RhaS plus CRP) was smaller (13-fold). Analysis of a plasmid library expressing alanine substitutions at every residue in the carboxyl-terminal domain of the α subunit (α-CTD) identified 15 residues (mostly in the DNA-binding determinant) that were important at both the full-length and truncated promoters. Only one substitution was defective at the full-length but not the truncated promoter, and this residue was located in the DNA-binding determinant. Six substitutions were defective only at the promoter activated by RhaS alone, and these may define a protein-contacting determinant on α-CTD. Overall, our results suggest that CRP interaction with α-CTD may not be required for rhaBAD activation; however, α-CTD does contribute to full activation, probably through interactions with DNA and possibly RhaS.

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 In vitro reconstitution of a heterotrimeric nucleoporin complex consisting of recombinant Nsp1p, Nup49p, and Nup57p.

1997 ◽  
Vol 8 (1) ◽  
pp. 33-46 ◽  
Author(s):  
N L Schlaich ◽  
M Häner ◽  
A Lustig ◽  
U Aebi ◽  
E C Hurt
Keyword(s):  
Nucleocytoplasmic Transport ◽  
Full Length ◽  
Yeast Cells ◽  
Heptad Repeat ◽  
Nuclear Pores ◽  
Terminal Domain ◽  
Carboxyl Terminal Domain ◽  
Carboxyl Terminal

The yeast nucleoporins Nsp1p, Nup49p, and Nup57p form a complex at the nuclear pores which is involved in nucleocytoplasmic transport. To investigate the molecular basis underlying complex formation, recombinant full-length Nup49p and Nup57p and the carboxyl-terminal domain of Nsp1p, which lacks the FXFG repeat domain, were expressed in Escherichia coli. When the three purified proteins were mixed together, they spontaneously associated to form a 150-kDa complex of 1:1:1 stoichiometry. In this trimeric complex, Nup57p fulfills the role of an organizing center, to which Nup49p and Nsp1p individually bind. For this interaction to occur, only two heptad repeat regions of the Nsp1p carboxyl-terminal domain are required, each region being about 50 amino acids in length. Finally, the reconstituted complex has the capability to bind to full-length Nic96p but not to mutant forms which also do not interact in vivo. When added to permeabilized yeast cells, the complex associates with the nuclear envelope and the nuclear pores. We conclude that Nsp1p, Nup49p, and Nup57p can reconstitute a complex in vitro which is competent for further assembly with other components of nuclear pores.

scholarly journals Campylobacter jejuni Type VI Secretion System: Roles in Adaptation to Deoxycholic Acid, Host Cell Adherence, Invasion, and In Vivo Colonization

PLoS ONE ◽  
2012 ◽  
Vol 7 (8) ◽  
pp. e42842 ◽  
Author(s):  
Kvin Lertpiriyapong ◽  
Eric R. Gamazon ◽  
Yan Feng ◽  
Danny S. Park ◽  
Jassia Pang ◽  
...  
Keyword(s):  
Host Cell ◽  
Campylobacter Jejuni ◽  
Deoxycholic Acid ◽  
Secretion System ◽  
Type Vi Secretion System ◽  
Cell Adherence ◽  
Type Vi Secretion

scholarly journals TssA from Burkholderia cenocepacia: expression, purification, crystallization and crystallographic analysis

2018 ◽  
Vol 74 (9) ◽  
pp. 536-542 ◽  
Author(s):  
Hayley J. Owen ◽  
Ruyue Sun ◽  
Asma Ahmad ◽  
Svetlana E. Sedelnikova ◽  
Patrick J. Baker ◽  
...  
Keyword(s):  
Phylogenetic Analysis ◽  
Structure Determination ◽  
Crystallographic Analysis ◽  
Secretion System ◽  
Burkholderia Cenocepacia ◽  
Type Vi Secretion System ◽  
Core Component ◽  
The Third ◽  
Terminal Domain ◽  
Type Vi Secretion

TssA is a core component of the type VI secretion system, and phylogenetic analysis of TssA subunits from different species has suggested that these proteins fall into three distinct clades. Whilst representatives of two clades, TssA1 and TssA2, have been the subjects of investigation, no members of the third clade (TssA3) have been studied. Constructs of TssA from Burkholderia cenocepacia, a representative of clade 3, were expressed, purified and subjected to crystallization trials. Data were collected from crystals of constructs of the N-terminal and C-terminal domains. Analysis of the data from the crystals of these constructs and preliminary structure determination indicates that the C-terminal domain forms an assembly of 32 subunits in D 16 symmetry, whereas the N-terminal domain is not involved in subunit assocation.

scholarly journals Cholesterol and host cell surface proteins contribute to cell-cell fusion induced by the Burkholderia type VI secretion system 5

PLoS ONE ◽  
2017 ◽  
Vol 12 (10) ◽  
pp. e0185715 ◽  
Author(s):  
Liam Whiteley ◽  
Maria Haug ◽  
Kristina Klein ◽  
Matthias Willmann ◽  
Erwin Bohn ◽  
...  
Keyword(s):  
Cell Surface ◽  
Host Cell ◽  
Cell Fusion ◽  
Secretion System ◽  
Surface Proteins ◽  
Type Vi Secretion System ◽  
Cell Surface Proteins ◽  
Type Vi Secretion ◽  
Host Cell Surface ◽  
Cell Cell

scholarly journals Identification of a new effector-immunity pair of Aeromonas hydrophila type VI secretion system

2020 ◽  
Vol 51 (1) ◽  
Author(s):  
Shuiyan Ma ◽  
Yuhao Dong ◽  
Nannan Wang ◽  
Jin Liu ◽  
Chengping Lu ◽  
...  
Keyword(s):  
Aeromonas Hydrophila ◽  
Secretion System ◽  
Type Vi Secretion System ◽  
Type Vi Secretion

scholarly journals N-Acylhomoserine lactones involved in quorum sensing control the type VI secretion system, biofilm formation, protease production, and in vivo virulence in a clinical isolate of Aeromonas hydrophila

Microbiology ◽  
2009 ◽  
Vol 155 (11) ◽  
pp. 3518-3531 ◽  
Author(s):  
Bijay K. Khajanchi ◽  
Jian Sha ◽  
Elena V. Kozlova ◽  
Tatiana E. Erova ◽  
Giovanni Suarez ◽  
...  
Keyword(s):  
Quorum Sensing ◽  
Aeromonas Hydrophila ◽  
Biofilm Formation ◽  
Secretion System ◽  
Protease Production ◽  
Type Vi Secretion System ◽  
Double Knockout ◽  
Electron Microscopic ◽  
Type Vi Secretion

In this study, we delineated the role of N-acylhomoserine lactone(s) (AHLs)-mediated quorum sensing (QS) in the virulence of diarrhoeal isolate SSU of Aeromonas hydrophila by generating a double knockout ΔahyRI mutant. Protease production was substantially reduced in the ΔahyRI mutant when compared with that in the wild-type (WT) strain. Importantly, based on Western blot analysis, the ΔahyRI mutant was unable to secrete type VI secretion system (T6SS)-associated effectors, namely haemolysin coregulated protein and the valine-glycine repeat family of proteins, while significant levels of these effectors were detected in the culture supernatant of the WT A. hydrophila. In contrast, the production and translocation of the type III secretion system (T3SS) effector AexU in human colonic epithelial cells were not affected when the ahyRI genes were deleted. Solid surface-associated biofilm formation was significantly reduced in the ΔahyRI mutant when compared with that in the WT strain, as determined by a crystal violet staining assay. Scanning electron microscopic observations revealed that the ΔahyRI mutant was also defective in the formation of structured biofilm, as it was less filamentous and produced a distinct exopolysaccharide on its surface when compared with the structured biofilm produced by the WT strain. These effects of AhyRI could be complemented either by expressing the ahyRI genes in trans or by the exogeneous addition of AHLs to the ΔahyRI/ahyR+ complemented strain. In a mouse lethality experiment, 50 % attenuation was observed when we deleted the ahyRI genes from the parental strain of A. hydrophila. Together, our data suggest that AHL-mediated QS modulates the virulence of A. hydrophila SSU by regulating the T6SS, metalloprotease production and biofilm formation.

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