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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Isolation protocol	First author	Year	EV-METRIC
	EV130025	2/2	Salmonella enterica	Bacteria	0.2 µm filter dUC Sucrose-DG (valid.)	Guidi R	2013	78%
Study summary Full title Salmonella enterica delivers its genotoxin through outer membrane vesicles secreted from infected cells 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 electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation 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 Focus vesicles OMV Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography 0.2 µm filter + dUC + Sucrose-DG (valid.) Adj. k-factor 602 (pelleting) / 602 (washing) Protein markers EV: None 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 Isolation Method Differential ultracentrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g 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 1 Density medium Sucrose 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 Detected contaminants Cell organelle protein Characterization: Particle analysis EM EM-type transmission EM Image type Close-up, Wide-field

	EV130025	1/2	Salmonella enterica	Cell culture supernatant	0.2 µm filter dUC	Guidi R	2013	22%
Study summary Full title Salmonella enterica delivers its genotoxin through outer membrane vesicles secreted from infected cells 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% (54th 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 electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation 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 Focus vesicles OMV Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography 0.2 µm filter + dUC 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 Isolation Method Differential ultracentrifugation 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: 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 Detected EV-associated proteins MHC1 Detected contaminants Cell organelle protein ELISA Detected EV-associated proteins MHC1 Characterization: Particle analysis EM EM-type transmission EM Image type Close-up

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