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You searched for: EV130025 (EV-TRACK ID)

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Experiment number
  • If needed, multiple experiments were identified in a single publication based on differing sample types, separation protocols and/or vesicle types of interest.
Species
  • Species of origin of the EVs.
Separation protocol
  • Gives a short, non-chronological overview of the different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
Experiment number
  • Experiments differ in Sample type
Experiment number
  • Experiments differ in Sample type
Details EV-TRACK ID Experiment nr. Species Sample type Separation protocol First author Year EV-METRIC
EV130025 2/2 Salmonella enterica Bacteria (d)(U)C
DG
Filtration
Guidi R 2013 78%

Study summary

Full title
All authors
Guidi R, Levi L, Rouf SF, Puiac S, Rhen M, Frisan T
Journal
Cell Microbiol
Abstract
Cytolethal-distending toxins (CDTs) belong to a family of DNA damage inducing exotoxins that are pro (show more...)Cytolethal-distending toxins (CDTs) belong to a family of DNA damage inducing exotoxins that are produced by several Gram-negative bacteria. Salmonella enterica serovar Typhi expresses its CDT (named as Typhoid toxin) only in the Salmonella-containing vacuole (SCV) of infected cells, which requires its export for cell intoxication. The mechanisms of secretion, release in the extracellular space and uptake by bystander cells are poorly understood. We have addressed these issues using a recombinant S. Typhimurium strain, MC71-CDT, where the genes encoding for the PltA, PltB and CdtB subunits of the Typhoid toxin are expressed under control of the endogenous promoters. MC71-CDT grown under conditions that mimic the SCV secreted the holotoxin in outer membrane vesicles (OMVs). Epithelial cells infected with MC71-CDT also secreted OMVs-like vesicles. The release of these extracellular vesicles required an intact SCV and relied on anterograde transport towards the cellular cortex on microtubule and actin tracks. Paracrine internalization of Typhoid toxin-loaded OMVs by bystander cells was dependent on dynamin-1, indicating active endocytosis. The subsequent induction of DNA damage required retrograde transport of the toxin through the Golgi complex. These data provide new insights on the mode of secretion of exotoxins by cells infected with intracellular bacteria. (hide)
EV-METRIC
78% (98th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Bacteria
Sample origin
NAY
Focus vesicles
OMV
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(d)(U)C
DG
Filtration
Adj. k-factor
602 (pelleting) / 602 (washing)
Protein markers
EV:
non-EV: Cell organelle protein
Proteomics
no
EV density (g/ml)
1.130
TEM measurements
50
Show all info
Study aim
Function
Sample
Species
Salmonella enterica
Sample Type
Bacteria
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Pelleting performed
Yes
Pelleting: time(min)
70
Pelleting: rotor type
TH641
Pelleting: adjusted k-factor
602.0
Wash: Rotor Type
TH641
Wash: adjusted k-factor
602.0
Density gradient
Only used for validation of main results
Yes
Lowest density fraction
0.2
Highest density fraction
2
Orientation
Bottom-up
Speed (g)
100000
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Western Blot
Antibody details provided?
No
Detected contaminants
Cell organelle protein
Characterization: Particle analysis
EM
EM-type
transmission EM
Image type
Close-up, Wide-field
Report size (nm)
50
EV130025 1/2 Salmonella enterica NAY (d)(U)C
Filtration
Guidi R 2013 22%

Study summary

Full title
All authors
Guidi R, Levi L, Rouf SF, Puiac S, Rhen M, Frisan T
Journal
Cell Microbiol
Abstract
Cytolethal-distending toxins (CDTs) belong to a family of DNA damage inducing exotoxins that are pro (show more...)Cytolethal-distending toxins (CDTs) belong to a family of DNA damage inducing exotoxins that are produced by several Gram-negative bacteria. Salmonella enterica serovar Typhi expresses its CDT (named as Typhoid toxin) only in the Salmonella-containing vacuole (SCV) of infected cells, which requires its export for cell intoxication. The mechanisms of secretion, release in the extracellular space and uptake by bystander cells are poorly understood. We have addressed these issues using a recombinant S. Typhimurium strain, MC71-CDT, where the genes encoding for the PltA, PltB and CdtB subunits of the Typhoid toxin are expressed under control of the endogenous promoters. MC71-CDT grown under conditions that mimic the SCV secreted the holotoxin in outer membrane vesicles (OMVs). Epithelial cells infected with MC71-CDT also secreted OMVs-like vesicles. The release of these extracellular vesicles required an intact SCV and relied on anterograde transport towards the cellular cortex on microtubule and actin tracks. Paracrine internalization of Typhoid toxin-loaded OMVs by bystander cells was dependent on dynamin-1, indicating active endocytosis. The subsequent induction of DNA damage required retrograde transport of the toxin through the Golgi complex. These data provide new insights on the mode of secretion of exotoxins by cells infected with intracellular bacteria. (hide)
EV-METRIC
22% (59th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
NAY
Focus vesicles
OMV
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(d)(U)C
Filtration
Adj. k-factor
602 (pelleting) / 602 (washing)
Protein markers
EV: MHC1
non-EV: Cell organelle protein
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Salmonella enterica
Sample Type
Cell culture supernatant
EV-harvesting Medium
EV Depleted
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 50,000 g and 100,000 g
Pelleting performed
Yes
Pelleting: time(min)
70
Pelleting: rotor type
TH641
Pelleting: adjusted k-factor
602.0
Wash: volume per pellet (ml)
10
Wash: Rotor Type
TH641
Wash: adjusted k-factor
602.0
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
MHC1
Detected contaminants
Cell organelle protein
ELISA
Antibody details provided?
No
Detected EV-associated proteins
MHC1
Characterization: Particle analysis
EM
EM-type
transmission EM
Image type
Close-up
1 - 2 of 2
  • CM = Commercial method
  • dUC = differential ultracentrifugation
  • DG = density gradient
  • UF = ultrafiltration
  • SEC = size-exclusion chromatography
EV-TRACK ID
EV130025
species
Salmonella enterica
sample type
Bacteria
Cell culture
cell type
NA
NAY
medium
EV Depleted
condition
NAY
NAY
separation protocol
(d)(U)C
DG
Filtration
(d)(U)C
Filtration
Exp. nr.
2
1
EV-METRIC %
78
22