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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV140050	1/1	Streptococcus pneumoniae	Bacteria	(d)(U)C DG Filtration SEC	Olaya-Abril A	2014	56%
Study summary Full title Characterization of protective extracellular membrane-derived vesicles produced by Streptococcus pneumoniae All authors Olaya-Abril A, Prados-Rosales R, McConnell MJ, Martín-Peña R, González-Reyes JA, Jiménez-Munguía I, Gómez-Gascón L, Fernández J, Luque-García JL, García-Lidón C, Estévez H, Pachón J, Obando I, Casadevall A, Pirofski LA, Rodríguez-Ortega MJ Journal J Proteomics Abstract Extracellular vesicles are produced by many pathogenic microorganisms and have varied functions that (show more...)Extracellular vesicles are produced by many pathogenic microorganisms and have varied functions that include secretion and release of microbial factors, which contribute to virulence. Very little is known about vesicle production by Gram-positive bacteria, as well as their biogenesis and release mechanisms. In this work, we demonstrate the active production of vesicles by Streptococcus pneumoniae from the plasma membrane, rather than being a product from cell lysis. We biochemically characterized them by proteomics and fatty acid analysis, showing that these vesicles and the plasma membrane resemble in essential aspects, but have some differences: vesicles are more enriched in lipoproteins and short-chain fatty acids. We also demonstrate that these vesicles act as carriers of surface proteins and virulence factors. They are also highly immunoreactive against human sera and induce immune responses that protect against infection. Overall, this work provides insights into the biology of this important Gram-positive human pathogen and the role of extracellular vesicles in clinical applications.BIOLOGICAL SIGNIFICANCE: Pneumococcus is one of the leading causes of bacterial pneumonia worldwide in children and the elderly, being responsible for high morbidity and mortality rates in developing countries. The augment of pneumococcal disease in developed countries has raised major public health concern, since the difficulties to treat these infections due to increasing antibiotic resistance. Vaccination is still the best way to combat pneumococcal infections. One of the mechanisms that bacterial pathogens use to combat the defense responses of invaded hosts is the production and release of extracellular vesicles derived from the outer surface. Little is known about this phenomenon in Gram-positives. We show that pneumococcus produces membrane-derived vesicles particularly enriched in lipoproteins. We also show the utility of pneumococcal vesicles as a new type of vaccine, as they induce protection in immunized mice against infection with a virulent strain. This work will contribute to understand the role of these structures in important biological processes such as host-pathogen interactions and prevention of human disease. (hide) EV-METRIC 56% (92nd 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 extracellular 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 DG Filtration SEC Protein markers EV: Pneumococcal surface protein A/ MalX non-EV: Pneumolysin Proteomics yes TEM measurements 20-75 Show all info Study aim Function Sample Species Streptococcus pneumoniae Sample Type Bacteria Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 90 Density gradient Only used for validation of main results Yes Lowest density fraction 5 Highest density fraction 35 Orientation Bottom-up Speed (g) 100000 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Western Blot Detected EV-associated proteins "MalX/ Pneumococcal surface protein A" Detected contaminants Pneumolysin ELISA Detected EV-associated proteins "MalX/ Pneumococcal surface protein A" Characterization: Particle analysis EM EM-type transmission EM/ immune EM/ scanning EM EM protein Pneumolysin Report size (nm) 20-75

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EV-TRACK ID	EV140050
species	Streptococcus pneumoniae
sample type	Bacteria
condition	NAY
separation protocol	(d)(U)C DG Filtration SEC
Exp. nr.	1
EV-METRIC %	56