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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV210136	1/2	Pseudomonas maltophilia	Pseudomonas maltophilia	(d)(U)C DG Filtration	Ferrer-Navarro, Mario	2016	28%
Study summary Full title Proteomic analysis of outer membrane proteins and vesicles of a clinical isolate and a collection strain of Stenotrophomonas maltophilia All authors Mario Ferrer-Navarro, Gerard Torrent, Elías Mongiardini, Oscar Conchillo-Solé, Isidre Gibert, Xavier Daura Journal J Proteomics Abstract Stenotrophomonas maltophilia is a Gram-negative pathogen with emerging nosocomial incidence that dis (show more...)Stenotrophomonas maltophilia is a Gram-negative pathogen with emerging nosocomial incidence that displays a high genomic diversity, complicating the study of its pathogenicity, virulence and resistance factors. The interaction of bacterial pathogens with host cells is largely mediated by outer membrane proteins (OMPs). Indeed, several OMPs of Gram-negative bacteria have been recognized as important virulence factors and targets for host immune recognition or to be involved in mechanisms of resistance to antimicrobials. OMPs are also present in outer membrane vesicles (OMVs), which bacteria constitutively secrete to the extracellular milieu and are essential for bacterial survival and pathogenesis. Here, we report the characterization of the OMP and native OMV subproteomes of a clinical isolate (M30) and a collection strain (ATCC13637) of S. maltophilia. We had previously shown that the ATCC13637 strain has an attenuated phenotype in a zebrafish model of infection, as well as a distinct susceptibility profile against a panel of antimicrobials. The protein profiles of the OMP and OMV subproteomes of these two strains and their differences consequently point at pathogenesis, virulence or resistance proteins, such as two variants of the quorum-sensing factor Ax21 that are found to be highly abundant in the OMP fraction and exported to OMVs. Biological significance: Stenotrophomonas maltophilia is rapidly climbing positions in the ranking of multidrug-resistant pathogens that are frequently isolated in hospital environments. Being an emerging human pathogen, the knowledge on the factors determining the pathogenicity, virulence and resistance traits of this microorganism is still scarce. Outer membrane proteins (OMPs) and vesicles (OMVs) are key elements for the interaction of Gram-negative bacteria with their environment -including the host-and have fundamental roles in both infection and resistance processes. The present study sets a first basis for a phenotype-dependent characterisation of the OMP subproteome of S. maltophilia and complements very recent work on the OMV subproteome of this species. The variability found among even two strains demonstrates once more that the analysis of genotypically and phenotypically distinct isolates under various conditions will be required before we can draw a significant picture of the OMP and OMV subproteomes of S. maltophilia. (hide) EV-METRIC 28% (64th 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 ATCC 13637 Focus vesicles 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 (d)(U)C DG Filtration Protein markers EV: None non-EV: None Proteomics yes EV density (g/ml) Not specified Show all info Study aim Identification of content (omics approaches) Sample Species Pseudomonas maltophilia Sample Type Cell culture supernatant EV-producing cells Pseudomonas maltophilia EV-harvesting Medium Not specified Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Equal to or above 150,000 g Pelleting performed Yes Pelleting: time(min) 180 Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 150000 Density gradient Type Discontinous Number of initial discontinuous layers 3 Lowest density fraction 0.6 M Highest density fraction 2.5 M Total gradient volume, incl. sample (mL) 5 Sample volume (mL) 1,25 Orientation Top-down Rotor type Not specified Speed (g) 200000 Duration (min) 1200 Fraction volume (mL) Not specified Fraction processing Centrifugation Pelleting: volume per fraction Not spec Pelleting: duration (min) 120 Pelleting: rotor type Not specified Pelleting: speed (g) 200000 Filtration steps 0.45µm > x > 0.22µm, No Characterization: Protein analysis Protein Concentration Method 2D-Quant kit Proteomics database No Characterization: Lipid analysis No EM EM-type Transmission-EM Image type Wide-field

	EV210136	2/2	Pseudomonas maltophilia	Pseudomonas maltophilia	(d)(U)C DG Filtration	Ferrer-Navarro, Mario	2016	28%
Study summary Full title Proteomic analysis of outer membrane proteins and vesicles of a clinical isolate and a collection strain of Stenotrophomonas maltophilia All authors Mario Ferrer-Navarro, Gerard Torrent, Elías Mongiardini, Oscar Conchillo-Solé, Isidre Gibert, Xavier Daura Journal J Proteomics Abstract Stenotrophomonas maltophilia is a Gram-negative pathogen with emerging nosocomial incidence that dis (show more...)Stenotrophomonas maltophilia is a Gram-negative pathogen with emerging nosocomial incidence that displays a high genomic diversity, complicating the study of its pathogenicity, virulence and resistance factors. The interaction of bacterial pathogens with host cells is largely mediated by outer membrane proteins (OMPs). Indeed, several OMPs of Gram-negative bacteria have been recognized as important virulence factors and targets for host immune recognition or to be involved in mechanisms of resistance to antimicrobials. OMPs are also present in outer membrane vesicles (OMVs), which bacteria constitutively secrete to the extracellular milieu and are essential for bacterial survival and pathogenesis. Here, we report the characterization of the OMP and native OMV subproteomes of a clinical isolate (M30) and a collection strain (ATCC13637) of S. maltophilia. We had previously shown that the ATCC13637 strain has an attenuated phenotype in a zebrafish model of infection, as well as a distinct susceptibility profile against a panel of antimicrobials. The protein profiles of the OMP and OMV subproteomes of these two strains and their differences consequently point at pathogenesis, virulence or resistance proteins, such as two variants of the quorum-sensing factor Ax21 that are found to be highly abundant in the OMP fraction and exported to OMVs. Biological significance: Stenotrophomonas maltophilia is rapidly climbing positions in the ranking of multidrug-resistant pathogens that are frequently isolated in hospital environments. Being an emerging human pathogen, the knowledge on the factors determining the pathogenicity, virulence and resistance traits of this microorganism is still scarce. Outer membrane proteins (OMPs) and vesicles (OMVs) are key elements for the interaction of Gram-negative bacteria with their environment -including the host-and have fundamental roles in both infection and resistance processes. The present study sets a first basis for a phenotype-dependent characterisation of the OMP subproteome of S. maltophilia and complements very recent work on the OMV subproteome of this species. The variability found among even two strains demonstrates once more that the analysis of genotypically and phenotypically distinct isolates under various conditions will be required before we can draw a significant picture of the OMP and OMV subproteomes of S. maltophilia. (hide) EV-METRIC 28% (64th 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 Clinical strain M30 Focus vesicles 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 (d)(U)C DG Filtration Protein markers EV: None non-EV: None Proteomics yes EV density (g/ml) Not specified Show all info Study aim Identification of content (omics approaches) Sample Species Pseudomonas maltophilia Sample Type Cell culture supernatant EV-producing cells Pseudomonas maltophilia EV-harvesting Medium Not specified Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Equal to or above 150,000 g Pelleting performed Yes Pelleting: time(min) 180 Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 150000 Density gradient Type Discontinous Number of initial discontinuous layers 3 Lowest density fraction 0.6 M Highest density fraction 2.5 M Total gradient volume, incl. sample (mL) 5 Sample volume (mL) 1,25 Orientation Top-down Rotor type Not specified Speed (g) 200000 Duration (min) 1200 Fraction volume (mL) Not specified Fraction processing Centrifugation Pelleting: volume per fraction Not spec Pelleting: duration (min) 120 Pelleting: rotor type Not specified Pelleting: speed (g) 200000 Filtration steps 0.45µm > x > 0.22µm, No Characterization: Protein analysis Protein Concentration Method 2D-Quant kit Proteomics database No Characterization: Lipid analysis No EM EM-type Transmission-EM Image type Wide-field

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EV-TRACK ID	EV210136
species	Pseudomonas maltophilia
sample type	Cell culture
cell type	Pseudomonas maltophilia
condition	ATCC 13637	Clinical strain M30
separation protocol	(d)(U)C DG Filtration	(d)(U)C DG Filtration
Exp. nr.	1	2
EV-METRIC %	28	28