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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV120084	1/2	Aggregatibacter actinomycetemcomitans	Bacteria	(d)(U)C DG Filtration	Rompikuntal PK	2012	33%
Study summary Full title Perinuclear localization of internalized outer membrane vesicles carrying active cytolethal distending toxin from Aggregatibacter actinomycetemcomitans All authors Rompikuntal PK, Thay B, Khan MK, Alanko J, Penttinen AM, Asikainen S, Wai SN, Oscarsson J Journal Infect Immun Abstract Aggregatibacter actinomycetemcomitans is implicated in aggressive forms of periodontitis. Similarly (show more...)Aggregatibacter actinomycetemcomitans is implicated in aggressive forms of periodontitis. Similarly to several other Gram-negative species, this organism produces and excretes a cytolethal distending toxin (CDT), a genotoxin associated with cell distention, G2 cell cycle arrest, and/or apoptosis in many mammalian cell types. In this study, we have identified A. actinomycetemcomitans outer membrane vesicles (OMVs) as a vehicle for simultaneous delivery of multiple proteins, including CDT, into human cells. The OMV proteins were internalized in both HeLa cells and human gingival fibroblasts (HGF) via a mechanism of OMV fusion with lipid rafts in the plasma membrane. The active toxin unit, CdtB, was localized inside the nucleus of the intoxicated cells, whereas OmpA and proteins detected using an antibody specific to whole A. actinomycetemcomitans serotype a cells had a perinuclear distribution. In accordance with a tight association of CdtB with OMVs, vesicles isolated from A. actinomycetemcomitans strain D7SS (serotype a), in contrast to OMVs from a D7SS cdtABC mutant, induced a cytolethal distending effect on HeLa and HGF cells, indicating that OMV-associated CDT was biologically active. Association of CDT with OMVs was also observed in A. actinomycetemcomitans isolates belonging to serotypes b and c, indicating that OMV-mediated release of CDT may be conserved in A. actinomycetemcomitans. Although the role of A. actinomycetemcomitans OMVs in periodontal disease has not yet been elucidated, our present data suggest that OMVs could deliver biologically active CDT and additional virulence factors into susceptible cells of the periodontium. (hide) EV-METRIC 33% (82nd 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 184.6 (pelleting) / 184.6 (washing) Protein markers EV: LtxA/ CdtA/ OmpA/ CdtB non-EV: DNaK Proteomics no Show all info Study aim Function Sample Species Aggregatibacter actinomycetemcomitans Sample Type Bacteria Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 50,000 g and 100,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type 70Ti Pelleting: adjusted k-factor 184.6 Wash: Rotor Type 70Ti Wash: adjusted k-factor 184.6 Density gradient Only used for validation of main results Yes Lowest density fraction 10 Highest density fraction 45 Orientation Bottom-up Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins CdtA/ CdtB/ OmpA/ LtxA Detected contaminants DNaK ELISA Antibody details provided? No Detected EV-associated proteins CdtA/ CdtB/ OmpA/ LtxA Characterization: Particle analysis EM EM-type atomic force EM Image type Wide-field

	EV120084	2/2	Aggregatibacter actinomycetemcomitans	Bacteria	(d)(U)C DG Filtration	Rompikuntal PK	2012	33%
Study summary Full title Perinuclear localization of internalized outer membrane vesicles carrying active cytolethal distending toxin from Aggregatibacter actinomycetemcomitans All authors Rompikuntal PK, Thay B, Khan MK, Alanko J, Penttinen AM, Asikainen S, Wai SN, Oscarsson J Journal Infect Immun Abstract Aggregatibacter actinomycetemcomitans is implicated in aggressive forms of periodontitis. Similarly (show more...)Aggregatibacter actinomycetemcomitans is implicated in aggressive forms of periodontitis. Similarly to several other Gram-negative species, this organism produces and excretes a cytolethal distending toxin (CDT), a genotoxin associated with cell distention, G2 cell cycle arrest, and/or apoptosis in many mammalian cell types. In this study, we have identified A. actinomycetemcomitans outer membrane vesicles (OMVs) as a vehicle for simultaneous delivery of multiple proteins, including CDT, into human cells. The OMV proteins were internalized in both HeLa cells and human gingival fibroblasts (HGF) via a mechanism of OMV fusion with lipid rafts in the plasma membrane. The active toxin unit, CdtB, was localized inside the nucleus of the intoxicated cells, whereas OmpA and proteins detected using an antibody specific to whole A. actinomycetemcomitans serotype a cells had a perinuclear distribution. In accordance with a tight association of CdtB with OMVs, vesicles isolated from A. actinomycetemcomitans strain D7SS (serotype a), in contrast to OMVs from a D7SS cdtABC mutant, induced a cytolethal distending effect on HeLa and HGF cells, indicating that OMV-associated CDT was biologically active. Association of CDT with OMVs was also observed in A. actinomycetemcomitans isolates belonging to serotypes b and c, indicating that OMV-mediated release of CDT may be conserved in A. actinomycetemcomitans. Although the role of A. actinomycetemcomitans OMVs in periodontal disease has not yet been elucidated, our present data suggest that OMVs could deliver biologically active CDT and additional virulence factors into susceptible cells of the periodontium. (hide) EV-METRIC 33% (82nd 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 184.6 (pelleting) / 184.6 (washing) Protein markers EV: LtxA/ CdtA/ OmpA/ CdtB non-EV: DNaK Proteomics no Show all info Study aim Function Sample Species Aggregatibacter actinomycetemcomitans Sample Type Bacteria Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 50,000 g and 100,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type 70Ti Pelleting: adjusted k-factor 184.6 Wash: Rotor Type 70Ti Wash: adjusted k-factor 184.6 Density gradient Only used for validation of main results Yes Lowest density fraction 10 Highest density fraction 45 Orientation Bottom-up Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins CdtA/ CdtB/ OmpA/ LtxA Detected contaminants DNaK ELISA Antibody details provided? No Detected EV-associated proteins CdtA/ CdtB/ OmpA/ LtxA Characterization: Particle analysis EM EM-type atomic force EM Image type Wide-field

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EV-TRACK ID	EV120084
species	Aggregatibacter actinomycetemcomitans
sample type	Bacteria
condition	NAY
separation protocol	(d)(U)C DG Filtration	(d)(U)C DG Filtration
Exp. nr.	1	2
EV-METRIC %	33	33