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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV200185	1/3	Staphylococcus aureus	S. aureus NCTC6571	DG (d)(U)C Filtration	Bitto, Natalie J.	2021	78%
Study summary Full title Staphylococcus aureus membrane vesicles contain immunostimulatory DNA, RNA and peptidoglycan that activate innate immune receptors and induce autophagy All authors Natalie J Bitto, Lesley Cheng, Ella L Johnston, Rishi Pathirana, Thanh Kha Phan, Ivan K H Poon, Neil M O'Brien-Simpson, Andrew F Hill, Timothy P Stinear, Maria Kaparakis-Liaskos Journal J Extracell Vesicles Abstract Gram-positive bacteria ubiquitously produce membrane vesicles (MVs), and although they contribute to (show more...)Gram-positive bacteria ubiquitously produce membrane vesicles (MVs), and although they contribute to biological functions, our knowledge regarding their composition and immunogenicity remains limited. Here we examine the morphology, contents and immunostimulatory functions of MVs produced by three Staphylococcus aureus strains; a methicillin resistant clinical isolate, a methicillin sensitive clinical isolate and a laboratory-adapted strain. We observed differences in the number and morphology of MVs produced by each strain and showed that they contain microbe-associated molecular patterns (MAMPs) including protein, nucleic acids and peptidoglycan. Analysis of MV-derived RNA indicated the presence of small RNA (sRNA). Furthermore, we detected variability in the amount and composition of protein, nucleic acid and peptidoglycan cargo carried by MVs from each S. aureus strain. S. aureus MVs activated Toll-like receptor (TLR) 2, 7, 8, 9 and nucleotide-binding oligomerization domain containing protein 2 (NOD2) signalling and promoted cytokine and chemokine release by epithelial cells, thus identifying that MV-associated MAMPs including DNA, RNA and peptidoglycan are detected by pattern recognition receptors (PRRs). Moreover, S. aureus MVs induced the formation of and colocalized with autophagosomes in epithelial cells, while inhibition of lysosomal acidification using bafilomycin A1 resulted in accumulation of autophagosomal puncta that colocalized with MVs, revealing the ability of the host to degrade MVs via autophagy. This study reveals the ability of DNA, RNA and peptidoglycan associated with MVs to activate PRRs in host epithelial cells, and their intracellular degradation via autophagy. These findings advance our understanding of the immunostimulatory roles of Gram-positive bacterial MVs in mediating pathogenesis, and their intracellular fate within the host. (hide) EV-METRIC 78% (97th 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 Other / Membrane vesicles (MVs) 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 Density gradient (Differential) (ultra)centrifugation Filtration Protein markers EV: S. aureus proteins non-EV: None Proteomics no EV density (g/ml) 1.069-1.119 Show all info Study aim Function Sample Species Staphylococcus aureus Sample Type Cell culture supernatant EV-producing cells S. aureus NCTC6571 EV-harvesting Medium Serum free medium Cell count 2330000000 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type P28S Pelleting: speed (g) 100000 Density gradient Type Discontinuous Number of initial discontinuous layers 6 Lowest density fraction 20% Highest density fraction 45% Total gradient volume, incl. sample (mL) 12 Sample volume (mL) 1.1 Orientation Bottom-up Rotor type SW 40 Ti Speed (g) 100000 Duration (min) 960 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 12ml Pelleting: duration (min) 120 Pelleting: rotor type SW 40 Ti Pelleting: speed (g) 100000 Pelleting-wash: volume per pellet (mL) 12 Pelleting-wash: duration (min) 120 Pelleting-wash: speed (g) SW 40 Ti Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method Fluorometric assay (e.g. Qubit, NanoOrange,...) Western Blot Antibody details provided? No Detected EV-associated proteins S. aureus proteins Characterization: RNA analysis RNA analysis Type Capillary electrophoresis (e.g. Bioanalyzer) Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 50-500 EV concentration Yes Particle yield particles per colony forming units (CFU) of bacteria;Yes, other: 1010000000000 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV200185	2/3	Staphylococcus aureus	S. aureus BPH2760	DG (d)(U)C Filtration	Bitto, Natalie J.	2021	78%
Study summary Full title Staphylococcus aureus membrane vesicles contain immunostimulatory DNA, RNA and peptidoglycan that activate innate immune receptors and induce autophagy All authors Natalie J Bitto, Lesley Cheng, Ella L Johnston, Rishi Pathirana, Thanh Kha Phan, Ivan K H Poon, Neil M O'Brien-Simpson, Andrew F Hill, Timothy P Stinear, Maria Kaparakis-Liaskos Journal J Extracell Vesicles Abstract Gram-positive bacteria ubiquitously produce membrane vesicles (MVs), and although they contribute to (show more...)Gram-positive bacteria ubiquitously produce membrane vesicles (MVs), and although they contribute to biological functions, our knowledge regarding their composition and immunogenicity remains limited. Here we examine the morphology, contents and immunostimulatory functions of MVs produced by three Staphylococcus aureus strains; a methicillin resistant clinical isolate, a methicillin sensitive clinical isolate and a laboratory-adapted strain. We observed differences in the number and morphology of MVs produced by each strain and showed that they contain microbe-associated molecular patterns (MAMPs) including protein, nucleic acids and peptidoglycan. Analysis of MV-derived RNA indicated the presence of small RNA (sRNA). Furthermore, we detected variability in the amount and composition of protein, nucleic acid and peptidoglycan cargo carried by MVs from each S. aureus strain. S. aureus MVs activated Toll-like receptor (TLR) 2, 7, 8, 9 and nucleotide-binding oligomerization domain containing protein 2 (NOD2) signalling and promoted cytokine and chemokine release by epithelial cells, thus identifying that MV-associated MAMPs including DNA, RNA and peptidoglycan are detected by pattern recognition receptors (PRRs). Moreover, S. aureus MVs induced the formation of and colocalized with autophagosomes in epithelial cells, while inhibition of lysosomal acidification using bafilomycin A1 resulted in accumulation of autophagosomal puncta that colocalized with MVs, revealing the ability of the host to degrade MVs via autophagy. This study reveals the ability of DNA, RNA and peptidoglycan associated with MVs to activate PRRs in host epithelial cells, and their intracellular degradation via autophagy. These findings advance our understanding of the immunostimulatory roles of Gram-positive bacterial MVs in mediating pathogenesis, and their intracellular fate within the host. (hide) EV-METRIC 78% (97th 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 Other / Membrane vesicles (MVs) 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 Density gradient (Differential) (ultra)centrifugation Filtration Protein markers EV: S. aureus proteins non-EV: None Proteomics no EV density (g/ml) 1.069-1.119 Show all info Study aim Function Sample Species Staphylococcus aureus Sample Type Cell culture supernatant EV-producing cells S. aureus BPH2760 EV-harvesting Medium Serum free medium Cell count 2000000000 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type P28S Pelleting: speed (g) 100000 Density gradient Type Discontinuous Number of initial discontinuous layers 6 Lowest density fraction 20% Highest density fraction 45% Total gradient volume, incl. sample (mL) 12 Sample volume (mL) 1.1 Orientation Bottom-up Rotor type SW 40 Ti Speed (g) 100000 Duration (min) 960 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 12ml Pelleting: duration (min) 120 Pelleting: rotor type SW 40 Ti Pelleting: speed (g) 100000 Pelleting-wash: volume per pellet (mL) 12 Pelleting-wash: duration (min) 120 Pelleting-wash: speed (g) SW 40 Ti Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method Fluorometric assay (e.g. Qubit, NanoOrange,...) Western Blot Antibody details provided? No Detected EV-associated proteins S. aureus proteins Characterization: RNA analysis RNA analysis Type Capillary electrophoresis (e.g. Bioanalyzer) Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 50-500 EV concentration Yes Particle yield particles per colony forming units (CFU) of bacteria;Yes, other: 28000000000 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV200185	3/3	Staphylococcus aureus	S. aureus BPH2900	DG (d)(U)C Filtration	Bitto, Natalie J.	2021	78%
Study summary Full title Staphylococcus aureus membrane vesicles contain immunostimulatory DNA, RNA and peptidoglycan that activate innate immune receptors and induce autophagy All authors Natalie J Bitto, Lesley Cheng, Ella L Johnston, Rishi Pathirana, Thanh Kha Phan, Ivan K H Poon, Neil M O'Brien-Simpson, Andrew F Hill, Timothy P Stinear, Maria Kaparakis-Liaskos Journal J Extracell Vesicles Abstract Gram-positive bacteria ubiquitously produce membrane vesicles (MVs), and although they contribute to (show more...)Gram-positive bacteria ubiquitously produce membrane vesicles (MVs), and although they contribute to biological functions, our knowledge regarding their composition and immunogenicity remains limited. Here we examine the morphology, contents and immunostimulatory functions of MVs produced by three Staphylococcus aureus strains; a methicillin resistant clinical isolate, a methicillin sensitive clinical isolate and a laboratory-adapted strain. We observed differences in the number and morphology of MVs produced by each strain and showed that they contain microbe-associated molecular patterns (MAMPs) including protein, nucleic acids and peptidoglycan. Analysis of MV-derived RNA indicated the presence of small RNA (sRNA). Furthermore, we detected variability in the amount and composition of protein, nucleic acid and peptidoglycan cargo carried by MVs from each S. aureus strain. S. aureus MVs activated Toll-like receptor (TLR) 2, 7, 8, 9 and nucleotide-binding oligomerization domain containing protein 2 (NOD2) signalling and promoted cytokine and chemokine release by epithelial cells, thus identifying that MV-associated MAMPs including DNA, RNA and peptidoglycan are detected by pattern recognition receptors (PRRs). Moreover, S. aureus MVs induced the formation of and colocalized with autophagosomes in epithelial cells, while inhibition of lysosomal acidification using bafilomycin A1 resulted in accumulation of autophagosomal puncta that colocalized with MVs, revealing the ability of the host to degrade MVs via autophagy. This study reveals the ability of DNA, RNA and peptidoglycan associated with MVs to activate PRRs in host epithelial cells, and their intracellular degradation via autophagy. These findings advance our understanding of the immunostimulatory roles of Gram-positive bacterial MVs in mediating pathogenesis, and their intracellular fate within the host. (hide) EV-METRIC 78% (97th 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 Other / Membrane vesicles (MVs) 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 Density gradient (Differential) (ultra)centrifugation Filtration Protein markers EV: S. aureus proteins non-EV: None Proteomics no EV density (g/ml) 1.069-1.119 Show all info Study aim Function Sample Species Staphylococcus aureus Sample Type Cell culture supernatant EV-producing cells S. aureus BPH2900 EV-harvesting Medium Serum free medium Cell count 2130000000 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type P28S Pelleting: speed (g) 100000 Density gradient Type Discontinuous Number of initial discontinuous layers 6 Lowest density fraction 20% Highest density fraction 45% Total gradient volume, incl. sample (mL) 12 Sample volume (mL) 1.1 Orientation Bottom-up Rotor type SW 40 Ti Speed (g) 100000 Duration (min) 960 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 12ml Pelleting: duration (min) 120 Pelleting: rotor type SW 40 Ti Pelleting: speed (g) 100000 Pelleting-wash: volume per pellet (mL) 12 Pelleting-wash: duration (min) 120 Pelleting-wash: speed (g) SW 40 Ti Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method Fluorometric assay (e.g. Qubit, NanoOrange,...) Western Blot Antibody details provided? No Detected EV-associated proteins S. aureus proteins Characterization: RNA analysis RNA analysis Type Capillary electrophoresis (e.g. Bioanalyzer) Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 50-500 EV concentration Yes Particle yield particles per colony forming units (CFU) of bacteria;Yes, other: 53000000000 EM EM-type Transmission-EM Image type Close-up, Wide-field

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EV-TRACK ID	EV200185
species	Staphylococcus aureus
sample type	Cell culture
cell type	S. aureus NCTC6571	S. aureus BPH2760	S. aureus BPH2900
condition	Control condition	Control condition	Control condition
separation protocol	Density gradient dUC Filtration	Density gradient dUC Filtration	Density gradient dUC Filtration
Exp. nr.	1	2	3
EV-METRIC %	78	78	78