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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV230891	1/1	Edwardsiella tarda	0909I strain	(d)(U)C DG Filtration UF	Chen S	2018	44%
Study summary Full title Dysregulated hemolysin liberates bacterial outer membrane vesicles for cytosolic lipopolysaccharide sensing. All authors Chen S, Yang D, Wen Y, Jiang Z, Zhang L, Jiang J, Chen Y, Hu T, Wang Q, Zhang Y, Liu Q Journal PLoS Pathog Abstract Inflammatory caspase-11/4/5 recognize cytosolic LPS from invading Gram-negative bacteria and induce (show more...)Inflammatory caspase-11/4/5 recognize cytosolic LPS from invading Gram-negative bacteria and induce pyroptosis and cytokine release, forming rapid innate antibacterial defenses. Since extracellular or vacuole-constrained bacteria are thought to rarely access the cytoplasm, how their LPS are exposed to the cytosolic sensors is a critical event for pathogen recognition. Hemolysin is a pore-forming bacterial toxin, which was generally accepted to rupture cell membrane, leading to cell lysis. Whether and how hemolysin participates in non-canonical inflammasome signaling remains undiscovered. Here, we show that hemolysin-overexpressed enterobacteria triggered significantly increased caspase-4 activation in human intestinal epithelial cell lines. Hemolysin promoted LPS cytosolic delivery from extracellular bacteria through dynamin-dependent endocytosis. Further, we revealed that hemolysin was largely associated with bacterial outer membrane vesicles (OMVs) and induced rupture of OMV-containing vacuoles, subsequently increasing LPS exposure to the cytosolic sensor. Accordingly, overexpression of hemolysin promoted caspase-11 dependent IL-18 secretion and gut inflammation in mice, which was associated with restricting bacterial colonization in vivo. Together, our work reveals a concept that hemolysin promotes noncanonical inflammasome activation via liberating OMVs for cytosolic LPS sensing, which offers insights into innate immune surveillance of dysregulated hemolysin via caspase-11/4 in intestinal antibacterial defenses. (hide) EV-METRIC 44% (84th 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 OMV (outer membrane vesicles) 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: OmpA/ EthA non-EV: RNAP Proteomics no Show all info Study aim Function Sample Species Edwardsiella tarda Sample Type Cell culture supernatant EV-producing cells 0909I strain EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Equal to or above 150,000 g Pelleting performed Yes Pelleting: time(min) 90 Pelleting: rotor type NVTTM65 Pelleting: speed (g) 284100 Density gradient Type Discontinuous Number of initial discontinuous layers 5 Lowest density fraction 20% Highest density fraction 45% Total gradient volume, incl. sample (mL) 11 mL Sample volume (mL) 2 mL Orientation Bottom-up Rotor type NVTTM65 Speed (g) 284000 Duration (min) 960 Fraction volume (mL) 1 mL Filtration steps 0.45µm > x > 0.22µm, Ultra filtration Cut-off size (kDa) 30 Membrane type NS Characterization: Protein analysis Protein Concentration Method Bradford Western Blot Detected EV-associated proteins OmpA/ EthA Not detected contaminants RNAP Characterization: RNA analysis RNA analysis Type (RT)(q)PCR Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis EM EM-type Transmission-EM Image type Close-up

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EV-TRACK ID	EV230891
species	Edwardsiella tarda
sample type	Cell culture
cell type	0909I strain
condition	Control condition
separation protocol	dUC/ Density gradient/ Filtration/ Ultrafiltration
Exp. nr.	1
EV-METRIC %	44