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 Burkholderia cenocepacia utilizes a type VI secretion system for bacterial competition

2019 ◽  
Vol 8 (7) ◽  
Author(s):  
Helena L. Spiewak ◽  
Sravanthi Shastri ◽  
Lili Zhang ◽  
Stephan Schwager ◽  
Leo Eberl ◽  
...  
Keyword(s):  
Secretion System ◽  
Burkholderia Cenocepacia ◽  
Type Vi Secretion System ◽  
Bacterial Competition ◽  
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 Burkholderia cenocepacia Type VI Secretion System Mediates Escape of Type II Secreted Proteins into the Cytoplasm of Infected Macrophages

PLoS ONE ◽  
2012 ◽  
Vol 7 (7) ◽  
pp. e41726 ◽  
Author(s):  
Roberto Rosales-Reyes ◽  
Daniel F. Aubert ◽  
Jennifer S. Tolman ◽  
Amal O. Amer ◽  
Miguel A. Valvano
Keyword(s):  
Secretion System ◽  
Burkholderia Cenocepacia ◽  
Secreted Proteins ◽  
Type Ii ◽  
Type Vi Secretion System ◽  
Type Vi Secretion

scholarly journals Host Adaptation Predisposes Pseudomonas aeruginosa to Type VI Secretion System-Mediated Predation by the Burkholderia cepacia Complex

2020 ◽  
Author(s):  
Andrew I Perault ◽  
Courtney E Chandler ◽  
David A Rasko ◽  
Robert K Ernst ◽  
Matthew C Wolfgang ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Burkholderia Cepacia ◽  
Secretion System ◽  
Burkholderia Cenocepacia ◽  
Burkholderia Cepacia Complex ◽  
Dependent Manner ◽  
Type Vi Secretion System ◽  
Secretion Systems ◽  
Type Vi Secretion

SUMMARYPseudomonas aeruginosa (Pa) and Burkholderia cepacia complex (Bcc) species are opportunistic lung pathogens of individuals with cystic fibrosis (CF). While Pa can initiate long-term infections in younger CF patients, Bcc infections only arise in teenagers and adults. Both Pa and Bcc use type VI secretion systems (T6SS) to mediate interbacterial competition. Here, we show that Pa isolates from teenage/adult CF patients, but not those from young CF patients, are outcompeted by the epidemic Bcc isolate Burkholderia cenocepacia strain AU1054 (BcAU1054) in a T6SS-dependent manner. The genomes of susceptible Pa isolates harbor T6SS-abrogating mutations, the repair of which, in some cases, rendered the isolates resistant. Moreover, seven of eight Bcc strains outcompeted Pa strains isolated from the same patients. Our findings suggest that certain mutations that arise as Pa adapts to the CF lung abrogate T6SS activity, making Pa and its human host susceptible to potentially fatal Bcc superinfection.

scholarly journals The Burkholderia cenocepacia type VI secretion system effector TecA is a virulence factor during lung infection

2021 ◽  
Author(s):  
Nicole A. Loeven ◽  
Andrew I. Perault ◽  
Peggy A. Cotter ◽  
Craig A. Hodges ◽  
Joseph D. Schwartzman ◽  
...  
Keyword(s):  
Weight Loss ◽  
Clinical Isolate ◽  
Virulence Factor ◽  
Lung Infection ◽  
Secretion System ◽  
Burkholderia Cenocepacia ◽  
Burkholderia Cepacia Complex ◽  
Type Vi Secretion System ◽  
Type Vi Secretion ◽  
Lung Infections

Burkholderia cenocepacia (Bc) is a member of the Burkholderia cepacia complex (Bcc), a group of bacteria with members responsible for causing lung infections in cystic fibrosis (CF) patients. The most severe outcome of Bcc infection in CF patients is cepacia syndrome, a disease characterized by necrotizing pneumonia with bacteremia and sepsis. Bc is strongly associated with cepacia syndrome making it one of the most virulent members of the Bcc. Mechanisms underlying the pathogenesis of Bc in lung infections and cepacia syndrome remain to be uncovered. Bc is primarily an intracellular pathogen, and encodes the type VI secretion system (T6SS) anti-host effector TecA, which is translocated into host cells. TecA is a deamidase that inactivates multiple Rho GTPases, including RhoA. Inactivation of RhoA by TecA triggers assembly of the pyrin inflammasome, leading to secretion of proinflammatory cytokines such as IL-1β from macrophages. Previous work with the Bc clinical isolate J2315 showed that TecA increases immunopathology during acute lung infection in C57BL/6 mice and suggested that this effector acts as a virulence factor by triggering assembly of the pyrin inflammasome. Here, we extend these results using a second Bc clinical isolate, AU1054, to demonstrate that TecA exacerbates weight loss and lethality during lung infection in C57BL/6 mice and CF mice. Unexpectedly, pyrin was dispensable for TecA virulence activity in both mouse infection models. Our findings establish that TecA is a Bc virulence factor that exacerbates lung inflammation, weight loss, and lethality in a mouse lung infection model.

scholarly journals The Burkholderia cenocepacia Type VI Secretion System Effector TecA Is a Virulence Factor in Mouse Models of Lung Infection

mBio ◽  
2021 ◽  
Author(s):  
Nicole A. Loeven ◽  
Andrew I. Perault ◽  
Peggy A. Cotter ◽  
Craig A. Hodges ◽  
Joseph D. Schwartzman ◽  
...  
Keyword(s):  
Animal Model ◽  
Mouse Models ◽  
Close Association ◽  
Lung Infection ◽  
Secretion System ◽  
Burkholderia Cenocepacia ◽  
Type Vi Secretion System ◽  
Content Type ◽  
Type Vi Secretion ◽  
Lung Infections

B. cenocepacia is often considered the most virulent species in the Bcc because of its close association with cepacia syndrome in addition to its capacity to cause chronic lung infections in CF patients (1). Prior to the current study, virulence factors of B. cenocepacia important for causing lethal disease had not been identified in a CF animal model of lung infection.

scholarly journals An IcmF Family Protein, ImpLM, Is an Integral Inner Membrane Protein Interacting with ImpKL, and Its Walker A Motif Is Required for Type VI Secretion System-Mediated Hcp Secretion in Agrobacterium tumefaciens

2009 ◽  
Vol 191 (13) ◽  
pp. 4316-4329 ◽  
Author(s):  
Lay-Sun Ma ◽  
Jer-Sheng Lin ◽  
Erh-Min Lai
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Membrane Protein ◽  
Secretion System ◽  
Type Vi Secretion System ◽  
Inner Membrane ◽  
Terminal Domain ◽  
Type Vi Secretion ◽  
Family Protein ◽  
Biochemical Fractionation ◽  
Inner Membrane Protein

ABSTRACT An intracellular multiplication F (IcmF) family protein is a conserved component of a newly identified type VI secretion system (T6SS) encoded in many animal and plant-associated Proteobacteria. We have previously identified ImpLM, an IcmF family protein that is required for the secretion of the T6SS substrate hemolysin-coregulated protein (Hcp) from the plant-pathogenic bacterium Agrobacterium tumefaciens. In this study, we characterized the topology of ImpLM and the importance of its nucleotide-binding Walker A motif involved in Hcp secretion from A. tumefaciens. A combination of β-lactamase-green fluorescent protein fusion and biochemical fractionation analyses revealed that ImpLM is an integral polytopic inner membrane protein comprising three transmembrane domains bordered by an N-terminal domain facing the cytoplasm and a C-terminal domain exposed to the periplasm. impLM mutants with substitutions or deletions in the Walker A motif failed to complement the impLM deletion mutant for Hcp secretion, which provided evidence that ImpLM may bind and/or hydrolyze nucleoside triphosphates to mediate T6SS machine assembly and/or substrate secretion. Protein-protein interaction and protein stability analyses indicated that there is a physical interaction between ImpLM and another essential T6SS component, ImpKL. Topology and biochemical fractionation analyses suggested that ImpKL is an integral bitopic inner membrane protein with an N-terminal domain facing the cytoplasm and a C-terminal OmpA-like domain exposed to the periplasm. Further comprehensive yeast two-hybrid assays dissecting ImpLM-ImpKL interaction domains suggested that ImpLM interacts with ImpKL via the N-terminal cytoplasmic domains of the proteins. In conclusion, ImpLM interacts with ImpKL, and its Walker A motif is required for its function in mediation of Hcp secretion from A. tumefaciens.

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.

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