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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV100010	1/1	Bacillus anthracis	Bacteria	(d)(U)C UF	Rivera J	2010	44%
Study summary Full title Bacillus anthracis produces membrane-derived vesicles containing biologically active toxins All authors Rivera J, Cordero RJ, Nakouzi AS, Frases S, Nicola A, Casadevall A Journal Proc Natl Acad Sci U S A Abstract Extracellular vesicle production is a ubiquitous process in Gram-negative bacteria, but little is kn (show more...)Extracellular vesicle production is a ubiquitous process in Gram-negative bacteria, but little is known about such process in Gram-positive bacteria. We report the isolation of extracellular vesicles from the supernatants of Bacillus anthracis, a Gram-positive bacillus that is a powerful agent for biological warfare. B. anthracis vesicles formed at the outer layer of the bacterial cell had double-membrane spheres and ranged from 50 to 150 nm in diameter. Immunoelectron microscopy with mAbs to protective antigen, lethal factor, edema toxin, and anthrolysin revealed toxin components and anthrolysin in vesicles, with some vesicles containing more than one toxin component. Toxin-containing vesicles were also visualized inside B. anthracis-infected macrophages. ELISA and immunoblot analysis of vesicle preparations confirmed the presence of B. anthracis toxin components. A mAb to protective antigen protected macrophages against vesicles from an anthrolysin-deficient strain, but not against vesicles from Sterne 34F2 and Sterne ?T strains, consistent with the notion that vesicles delivered both toxin and anthrolysin to host cells. Vesicles were immunogenic in BALB/c mice, which produced a robust IgM response to toxin components. Furthermore, vesicle-immunized mice lived significantly longer than controls after B. anthracis challenge. Our results indicate that toxin secretion in B. anthracis is, at least, partially vesicle-associated, thus allowing concentrated delivery of toxin components to target host cells, a mechanism that may increase toxin potency. Our observations may have important implications for the design of vaccines, for passive antibody strategies, and provide a previously unexplored system for studying secretory pathways in Gram-positive bacteria. (hide) EV-METRIC 44% (90th 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 Membrane(-derived) 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 (d)(U)C UF Protein markers EV: Anthrax toxin polypeptides non-EV: Proteomics yes TEM measurements 50-200 Show all info Study aim Omics Sample Species Bacillus anthracis Sample Type Bacteria Separation Method (Differential) (ultra)centrifugation 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 performed Yes Pelleting: time(min) 60 Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins "Anthrax toxin polypeptides" ELISA Antibody details provided? No Detected EV-associated proteins "Anthrax toxin polypeptides" Flow cytometry specific beads Antibody details provided? No Antibody dilution provided? No Selected surface protein(s) Yes Characterization: Particle analysis DLS EM EM-type transmission EM/ immune EM EM protein Anthrax toxin polypeptides Image type Close-up, Wide-field Report size (nm) 50-200

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EV-TRACK ID	EV100010
species	Bacillus anthracis
sample type	Bacteria
condition	NAY
separation protocol	(d)(U)C UF
Exp. nr.	1
EV-METRIC %	44