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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV230674	1/2	Klebsiella pneumoniae	ATCC 43816	(d)(U)C DG Filtration UF	Cahill BK	2015	38%
Study summary Full title Klebsiella pneumoniae O antigen loss alters the outer membrane protein composition and the selective packaging of proteins into secreted outer membrane vesicles. All authors Cahill BK, Seeley KW, Gutel D, Ellis TN Journal Microbiol Res Abstract Klebsiella pneumoniae is a nosocomial pathogen which naturally secretes lipopolysaccharide (LPS) and (show more...)Klebsiella pneumoniae is a nosocomial pathogen which naturally secretes lipopolysaccharide (LPS) and cell envelope associated proteins into the environment through the production of outer membrane vesicles (OMVs). The loss of the LPS O antigen has been demonstrated in other bacterial species to significantly alter the composition of OMVs. Therefore, this study aimed to comprehensively analyze the impact of O antigen loss on the sub-proteomes of both the outer membrane and secreted OMVs from K. pneumoniae. As determined by LC-MS/MS, OMVs were highly enriched with outer membrane proteins involved in cell wall, membrane, and envelope biogenesis as compared to the source cellular outer membrane. Deletion of wbbO, the enzyme responsible for O antigen attachment to LPS, decreased but did not eliminate this enrichment effect. Additionally, loss of O antigen resulted in OMVs with increased numbers of proteins involved in post-translational modification, protein turnover, and chaperones as compared to secreted vesicles from the wild type. This alteration of OMV composition may be a compensatory mechanism to deal with envelope stress. This comprehensive analysis confirms the highly distinct protein composition of OMVs as compared to their source membrane, and provides evidence for a selective sorting mechanism that involves LPS polysaccharides. These data support the hypothesis that modifications to LPS alters both the mechanics of protein sorting and the contents of secreted OMVs and significantly impacts the protein composition of the outer membrane. (hide) EV-METRIC 38% (79th 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 Control condition Focus vesicles Outer membrane vesicle 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 (Differential) (ultra)centrifugation Density gradient Filtration Ultrafiltration Protein markers EV: GroEL non-EV: RecA Proteomics yes Show all info Study aim Function/Identification of content (omics approaches) Sample Species Klebsiella pneumoniae Sample Type Cell culture supernatant EV-producing cells ATCC 43816 EV-harvesting Medium serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Pelleting performed No Density gradient Type Discontinuous Number of initial discontinuous layers 6 Lowest density fraction 20% Highest density fraction 45% Orientation Bottom-up Speed (g) 100000 Fraction volume (mL) 1 Fraction processing None Filtration steps > 0.45 µm, 0.45µm > x > 0.22µm, Ultra filtration Cut-off size (kDa) 100 Membrane type regenerated cellulose Characterization: Protein analysis Protein Concentration Method Bradford Western Blot Detected EV-associated proteins GroEL Not detected contaminants RecA Proteomics database No Characterization: Lipid analysis Yes

	EV230674	2/2	Klebsiella pneumoniae	ATCC 43816	(d)(U)C DG Filtration UF	Cahill BK	2015	38%
Study summary Full title Klebsiella pneumoniae O antigen loss alters the outer membrane protein composition and the selective packaging of proteins into secreted outer membrane vesicles. All authors Cahill BK, Seeley KW, Gutel D, Ellis TN Journal Microbiol Res Abstract Klebsiella pneumoniae is a nosocomial pathogen which naturally secretes lipopolysaccharide (LPS) and (show more...)Klebsiella pneumoniae is a nosocomial pathogen which naturally secretes lipopolysaccharide (LPS) and cell envelope associated proteins into the environment through the production of outer membrane vesicles (OMVs). The loss of the LPS O antigen has been demonstrated in other bacterial species to significantly alter the composition of OMVs. Therefore, this study aimed to comprehensively analyze the impact of O antigen loss on the sub-proteomes of both the outer membrane and secreted OMVs from K. pneumoniae. As determined by LC-MS/MS, OMVs were highly enriched with outer membrane proteins involved in cell wall, membrane, and envelope biogenesis as compared to the source cellular outer membrane. Deletion of wbbO, the enzyme responsible for O antigen attachment to LPS, decreased but did not eliminate this enrichment effect. Additionally, loss of O antigen resulted in OMVs with increased numbers of proteins involved in post-translational modification, protein turnover, and chaperones as compared to secreted vesicles from the wild type. This alteration of OMV composition may be a compensatory mechanism to deal with envelope stress. This comprehensive analysis confirms the highly distinct protein composition of OMVs as compared to their source membrane, and provides evidence for a selective sorting mechanism that involves LPS polysaccharides. These data support the hypothesis that modifications to LPS alters both the mechanics of protein sorting and the contents of secreted OMVs and significantly impacts the protein composition of the outer membrane. (hide) EV-METRIC 38% (79th 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 wbbO K.O. Focus vesicles Outer membrane vesicle 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 (Differential) (ultra)centrifugation Density gradient Filtration Ultrafiltration Protein markers EV: GroEL non-EV: RecA Proteomics yes Show all info Study aim Function/Identification of content (omics approaches) Sample Species Klebsiella pneumoniae Sample Type Cell culture supernatant EV-producing cells ATCC 43816 EV-harvesting Medium serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Pelleting performed No Density gradient Type Discontinuous Number of initial discontinuous layers 6 Lowest density fraction 20% Highest density fraction 45% Orientation Bottom-up Speed (g) 100000 Fraction volume (mL) 1 Fraction processing None Filtration steps > 0.45 µm, 0.45µm > x > 0.22µm, Ultra filtration Cut-off size (kDa) 100 Membrane type regenerated cellulose Characterization: Protein analysis Protein Concentration Method Bradford Western Blot Detected EV-associated proteins GroEL Not detected contaminants RecA Proteomics database No Characterization: Lipid analysis Yes

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EV-TRACK ID	EV230674
species	Klebsiella pneumoniae
sample type	Cell culture
cell type	ATCC 43816
condition	Control condition	wbbO K.O.
separation protocol	dUC/ Density gradient/ Filtration/ Ultrafiltration	dUC/ Density gradient/ Filtration/ Ultrafiltration
Exp. nr.	1	2
EV-METRIC %	38	38