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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV120175	1/1	Escherichia coli	Bacteria	(d)(U)C Filtration	Yu H	2012	0%
Study summary Full title YgfZ contributes to secretion of cytotoxic necrotizing factor 1 into outer-membrane vesicles in Escherichia coli All authors Yu H, Kim KS Journal Microbiology Abstract Cytotoxic necrotizing factor 1 (CNF1), a Rho GTPase-activating bacterial toxin, has been shown to co (show more...)Cytotoxic necrotizing factor 1 (CNF1), a Rho GTPase-activating bacterial toxin, has been shown to contribute to invasion by meningitis-causing Escherichia coli K1 of human brain microvascular endothelial cells (HBMEC), which constitute the blood-brain barrier. However, CNF1 is a cytosolic protein and it remains unclear how its secretion occurs, contributing to E. coli invasion of HBMEC. To investigate the genetic requirement for CNF1 secretion in E. coli K1 strain RS218, we performed mini-Tn5 in vitro mutagenesis and constructed a transposon mutant library of strain NBC, in which ?-lactamase was fused to the C-terminus of CNF1 in the chromosome of strain RS218. We identified a transposon mutant (NBC-1E6) that exhibited reduced ?-lactamase activity in its culture supernatant and had the transposon inserted into the ygfZ gene. When ygfZ was deleted from the genome of strain RS218 (?ygfZ), the translocation of CNF1 into HBMEC was impaired. Subcellular localization analysis of CNF1 demonstrated that YgfZ, a periplasmic protein, contributes to secretion of CNF1 into outer-membrane vesicles (OMVs). The ?ygfZ mutant was significantly defective in invasion of HBMEC compared to the parent E. coli K1 strain. The defects of the ?ygfZ mutant in CNF1 secretion into OMVs and translocation into HBMEC as well as invasion of HBMEC were abrogated by complementation with ygfZ. Taken together, our findings demonstrate that YgfZ contributes to CNF1 secretion into OMVs in meningitis-causing E. coli K1. (hide) EV-METRIC 0% (median: 14% 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 OMV 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 Filtration Protein markers EV: OmpA non-EV: Proteomics no Show all info Study aim Biogenesis/Sorting Sample Species Escherichia coli Sample Type Bacteria 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) 180 Filtration steps 0.45µm > x > 0.22µm, Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins OmpA ELISA Antibody details provided? No Detected EV-associated proteins OmpA

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EV-TRACK ID	EV120175
species	Escherichia coli
sample type	Bacteria
condition	NAY
separation protocol	(d)(U)C Filtration
Exp. nr.	1
EV-METRIC %	0