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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV100027	2/2	Leishmania donovani Leishmania mexicana Leishmania major	NAY	(d)(U)C DG	Silverman JM	2010	67%
Study summary Full title An exosome-based secretion pathway is responsible for protein export from Leishmania and communication with macrophages All authors Silverman JM, Clos J, de'Oliveira CC, Shirvani O, Fang Y, Wang C, Foster LJ, Reiner NE Journal J Cell Sci Abstract Specialized secretion systems are used by numerous bacterial pathogens to export virulence factors i (show more...)Specialized secretion systems are used by numerous bacterial pathogens to export virulence factors into host target cells. Leishmania and other eukaryotic intracellular pathogens also deliver effector proteins into host cells; however, the mechanisms involved have remained elusive. In this report, we identify exosome-based secretion as a general mechanism for protein secretion by Leishmania, and show that exosomes are involved in the delivery of proteins into host target cells. Comparative quantitative proteomics unambiguously identified 329 proteins in Leishmania exosomes, accounting for >52% of global protein secretion from these organisms. Our findings demonstrate that infection-like stressors (37 degrees C +/- pH 5.5) upregulated exosome release more than twofold and also modified exosome protein composition. Leishmania exosomes and exosomal proteins were detected in the cytosolic compartment of infected macrophages and incubation of macrophages with exosomes selectively induced secretion of IL-8, but not TNF-alpha. We thus provide evidence for an apparently broad-based mechanism of protein export by Leishmania. Moreover, we describe a mechanism for the direct delivery of Leishmania molecules into macrophages. These findings suggest that, like mammalian exosomes, Leishmania exosomes function in long-range communication and immune modulation. (hide) EV-METRIC 67% (94th 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 NAY Focus vesicles exosomes 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 Adj. k-factor 54.89 (pelleting) Protein markers EV: HSP90/ HSP70/ EF1A non-EV: Proteomics no EV density (g/ml) 1.08-1.17 Show all info Study aim Function Sample Species Leishmania donovani / Leishmania mexicana / Leishmania major Sample Type Cell culture supernatant EV-harvesting Medium serum free Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type TLA100.3 Pelleting: adjusted k-factor 54.89 Density gradient Only used for validation of main results Yes Lowest density fraction 0.25 Highest density fraction 2.5 Orientation Bottom-up Rotor type TLA100.3 Speed (g) 110000 Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins HSP90/ HSP70/ EF1A ELISA Antibody details provided? No Detected EV-associated proteins EF1A Characterization: Particle analysis EM EM-type transmission EM/ immune EM EM protein Hsp70 Image type Close-up, Wide-field

	EV100027	1/2	Leishmania donovani Leishmania mexicana Leishmania major	NAY	(d)(U)C DC UF	Silverman JM	2010	33%
Study summary Full title An exosome-based secretion pathway is responsible for protein export from Leishmania and communication with macrophages All authors Silverman JM, Clos J, de'Oliveira CC, Shirvani O, Fang Y, Wang C, Foster LJ, Reiner NE Journal J Cell Sci Abstract Specialized secretion systems are used by numerous bacterial pathogens to export virulence factors i (show more...)Specialized secretion systems are used by numerous bacterial pathogens to export virulence factors into host target cells. Leishmania and other eukaryotic intracellular pathogens also deliver effector proteins into host cells; however, the mechanisms involved have remained elusive. In this report, we identify exosome-based secretion as a general mechanism for protein secretion by Leishmania, and show that exosomes are involved in the delivery of proteins into host target cells. Comparative quantitative proteomics unambiguously identified 329 proteins in Leishmania exosomes, accounting for >52% of global protein secretion from these organisms. Our findings demonstrate that infection-like stressors (37 degrees C +/- pH 5.5) upregulated exosome release more than twofold and also modified exosome protein composition. Leishmania exosomes and exosomal proteins were detected in the cytosolic compartment of infected macrophages and incubation of macrophages with exosomes selectively induced secretion of IL-8, but not TNF-alpha. We thus provide evidence for an apparently broad-based mechanism of protein export by Leishmania. Moreover, we describe a mechanism for the direct delivery of Leishmania molecules into macrophages. These findings suggest that, like mammalian exosomes, Leishmania exosomes function in long-range communication and immune modulation. (hide) EV-METRIC 33% (75th 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 NAY Focus vesicles exosomes 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 DC UF Adj. k-factor 54.89 (pelleting) Protein markers EV: HSP90/ HSP70/ EF1A non-EV: Proteomics yes Show all info Study aim Function Sample Species Leishmania donovani / Leishmania mexicana / Leishmania major Sample Type Cell culture supernatant EV-harvesting Medium serum free Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type TLA100.3 Pelleting: adjusted k-factor 54.89 Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins HSP90/ HSP70/ EF1A ELISA Antibody details provided? No Detected EV-associated proteins EF1A Characterization: Particle analysis None

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EV-TRACK ID	EV100027
species	Leishmania donovani Leishmania mexicana Leishmania major
sample type	Cell culture
cell type	NAY
condition	NAY
separation protocol	(d)(U)C DG	(d)(U)C DC UF
Exp. nr.	2	1
EV-METRIC %	67	33