Summary of Study ST002130

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR001349. The data can be accessed directly via it's Project DOI: 10.21228/M8BM53 This work is supported by NIH grant, U2C- DK119886.

See: https://www.metabolomicsworkbench.org/about/howtocite.php

This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.

Study ID	ST002130
Study Title	Discovery and characterization of virulence associated functional metabolites in Escherichia coli based on functional metabolomics strategy（pellets metabolomics-1）
Study Summary	Bacterial metabolites are substrates of virulence factors of uropathogenic Escherichia coli (UPEC), but the mechanism underlying the role of functional metabolites in bacterial virulence from the perspective of small molecular metabolism is unclear. In the present study, we used a strategy of functional metabolomics integrated with bacterial genetics in attempt to decipher the mechanism of virulence formation in Escherichia coli (E. coli) from the viewpoint of small molecule metabolism. We identified the virulence-associated metabolome via analysis of the primary metabolome of the pathogenic UTI89 strain and the non-pathogenic MG1655 strain. Then, the iron-mediated virulence associated metabolome was identified by an iron fishing strategy. Also, the mechanism of siderophores in regulating pathogenicity in different environments was explored by investigating the effect of iron on siderophore biosynthesis. Finally, by knocking out genes related to siderophore biosynthesis, siderophore transport and iron utilization, siderophores dependent iron-regulating virulence associated metabolome, including 18 functional metabolites, was identified and verified to be involved in the regulation of bacterial virulence. Based on this we found that these functional metabolites regulated the virulence of E. coli by targeting multiple metabolic pathways in an iron-siderophores dependent manner. Moreover, a quantitative proteomics approach was implemented to further elucidate the mechanism of functional metabolites and functional proteins in modulating bacterial virulence. And our findings demonstrated that functional proteins regulated the virulence of E. coli by mediating iron binding, iron-siderophore transmembrane transport, and the biosynthesis and expression of functional metabolites. Interestingly, we found that functional metabolites enhance the virulence of E. coli by specifically modulating the key metabolic pathways involved in purine metabolism, proline metabolism, arginine metabolism and pyrimidine metabolism. Taken together, our study identified for the first time 18 functional metabolites regulating the of E. coli virulence, greatly enriching our understanding of the mechanism of functional metabolites that regulate the E. coli virulence by targeting primary metabolism, which will largely contribute to the development of new strategies to target virulence-based diagnosis and therapy of infections caused by different pathogens.
Institute	Shanghai Center for Systems Biomedicine, Shanghai Jiaotong University
Last Name	Lu
First Name	Haitao
Address	800 Dongchuan RD. Minhang District, Shanghai, Shanghai, 200240, China
Email	haitao.lu@sjtu.edu.cn
Phone	15221478139
Submit Date	2022-03-25
Raw Data Available	Yes
Raw Data File Type(s)	d
Analysis Type Detail	LC-MS
Release Date	2022-04-20
Release Version	1

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Project:

Project ID:	PR001349
Project DOI:	doi: 10.21228/M8BM53
Project Title:	Discovery and characterization of virulence associated functional metabolites in Escherichia coli based on functional metabolomics strategy
Project Type:	Untargeted MS quantitative analysis
Project Summary:	Discovery and characterization of virulence associated functional metabolites in Escherichia coli based on functional metabolomics strategy
Institute:	Shanghai Center for Systems Biomedicine, Shanghai Jiaotong University
Department:	Shanghai Center for Systems Biomedicine
Laboratory:	Lu Group
Last Name:	Lu
First Name:	Haitao
Address:	800 Dongchuan RD. Minhang District, Shanghai, Shanghai, 200240, China
Email:	longlonghu126@sjtu.edu.cn
Phone:	15221478139

Subject:

Subject ID:	SU002215
Subject Type:	Bacteria
Subject Species:	Escherichia coli
Taxonomy ID:	562

Factors:

Subject type: Bacteria; Subject species: Escherichia coli (Factor headings shown in green)

mb_sample_id	local_sample_id	Treatment
SA204464	WT-10-2P	10 μM iron supplementation
SA204465	WT-10-4P	10 μM iron supplementation
SA204466	WT-10-6P	10 μM iron supplementation
SA204467	WT-10-1P	10 μM iron supplementation
SA204468	WT-10-5P	10 μM iron supplementation
SA204469	WT-10-3P	10 μM iron supplementation
SA204470	entB-0-5P	standard growth conditions
SA204471	entB-0-6P	standard growth conditions
SA204472	iroA-0-1P	standard growth conditions
SA204473	iroA-0-2P	standard growth conditions
SA204474	entB-0-4P	standard growth conditions
SA204475	BS-0-6P	standard growth conditions
SA204476	iroA-0-3P	standard growth conditions
SA204477	entB-0-2P	standard growth conditions
SA204478	entB-0-1P	standard growth conditions
SA204479	entB-0-3P	standard growth conditions
SA204480	irp1-0-1P	standard growth conditions
SA204481	irp1-0-4P	standard growth conditions
SA204482	irp1-0-5P	standard growth conditions
SA204483	irp1-0-6P	standard growth conditions
SA204484	irp1-0-3P	standard growth conditions
SA204485	irp1-0-2P	standard growth conditions
SA204486	iroA-0-5P	standard growth conditions
SA204487	iroA-0-6P	standard growth conditions
SA204488	BS-0-5P	standard growth conditions
SA204489	iroA-0-4P	standard growth conditions
SA204490	BS-0-1P	standard growth conditions
SA204491	MG1655-0-1P	standard growth conditions
SA204492	MG1655-0-2P	standard growth conditions
SA204493	MG1655-0-3P	standard growth conditions
SA204494	MG1655-0-4P	standard growth conditions
SA204495	WT-0-6P	standard growth conditions
SA204496	WT-0-5P	standard growth conditions
SA204497	WT-0-2P	standard growth conditions
SA204498	WT-0-3P	standard growth conditions
SA204499	WT-0-4P	standard growth conditions
SA204500	MG1655-0-5P	standard growth conditions
SA204501	MG1655-0-6P	standard growth conditions
SA204502	ybtS-0-6P	standard growth conditions
SA204503	WT-0-1P	standard growth conditions
SA204504	BS-0-2P	standard growth conditions
SA204505	BS-0-3P	standard growth conditions
SA204506	ybtS-0-5P	standard growth conditions
SA204507	ybtS-0-4P	standard growth conditions
SA204508	ybtS-0-1P	standard growth conditions
SA204509	ybtS-0-2P	standard growth conditions
SA204510	ybtS-0-3P	standard growth conditions
SA204511	BS-0-4P	standard growth conditions

Showing results 1 to 48 of 48

Showing results 1 to 48 of 48

Collection:

Collection ID:	CO002208
Collection Summary:	After 18h of culture, the sample pellet was isolated. The bacterial pellets harvested from 50 mL of broth culture were mixed with 1.2 mL 80% ice-cold methanol (added to internal standard 0.001mg/ml 4-chloro-DL-phenylalanine), then vortexed for 30 s, and placed on dry ice for 30 min. The samples were centrifuged at 18000 × g for 15 min at 4 °C. The frozen samples were ground with beads and the homogenates were centrifuged at 18000 × g for 15 min at 4 °C. The supernatant was mixed with 800μL ice-cold acetonitrile, and then left to stand for 20 minutes in an ice bath. After centrifugation at 18000 × g 4℃ for 15 min, the supernatant was removed and filtered through 0.22μm membrane.
Sample Type:	Bacterial cells

Treatment:

Treatment ID:	TR002227
Treatment Summary:	M63 medium (1.36% monopotassium phosphate, 0.2% ammonium sulfate, 0.024% magnesium sulfate, 0.001% calcium chloride, and 0.0015% nicotinic acid) was used to form MG1655 and UTI89 mutants. In addition, add ferric chloride solution to the medium to prepare 10μM iron M63 medium, we cultured the wild UTI89 strain in the presence of 10μM iron. The E. coli strain was incubated in LB-agar plate for 12 hours, one colony was isolated to LB broth for further 4 hours incubation, then diluted the solution into M63 medium at a ratio of 1:100 and the cultures were incubated for another18 h at 37°C, 200rpm to culture E. coli.

Treatment ID:

TR002227

Treatment Summary:

M63 medium (1.36% monopotassium phosphate, 0.2% ammonium sulfate, 0.024% magnesium sulfate, 0.001% calcium chloride, and 0.0015% nicotinic acid) was used to form MG1655 and UTI89 mutants. In addition, add ferric chloride solution to the medium to prepare 10μM iron M63 medium, we cultured the wild UTI89 strain in the presence of 10μM iron. The E. coli strain was incubated in LB-agar plate for 12 hours, one colony was isolated to LB broth for further 4 hours incubation, then diluted the solution into M63 medium at a ratio of 1:100 and the cultures were incubated for another18 h at 37°C, 200rpm to culture E. coli.

Sample Preparation:

Sampleprep ID:	SP002221
Sampleprep Summary:	The bacterial pellets harvested from 50 mL of broth culture were mixed with 1.2 mL 80% ice-cold methanol (added to internal standard 0.001mg/ml 4-chloro-DL-phenylalanine), then vortexed for 30 s, and placed on dry ice for 30 min. The samples were centrifuged at 18000 × g for 15 min at 4 °C. The frozen samples were ground with beads and the homogenates were centrifuged at 18000 × g for 15 min at 4 °C. The supernatant was mixed with 800μL ice-cold acetonitrile, and then left to stand for 20 minutes in an ice bath. After centrifugation at 18000 × g 4℃ for 15 min, the supernatant was removed and filtered through 0.22μm membrane. For LC/MS based metabolomics analysis, the dried samples were dissolved in 200μL water and 5μL aliquots were analyzed.

Sampleprep ID:

SP002221

Sampleprep Summary:

The bacterial pellets harvested from 50 mL of broth culture were mixed with 1.2 mL 80% ice-cold methanol (added to internal standard 0.001mg/ml 4-chloro-DL-phenylalanine), then vortexed for 30 s, and placed on dry ice for 30 min. The samples were centrifuged at 18000 × g for 15 min at 4 °C. The frozen samples were ground with beads and the homogenates were centrifuged at 18000 × g for 15 min at 4 °C. The supernatant was mixed with 800μL ice-cold acetonitrile, and then left to stand for 20 minutes in an ice bath. After centrifugation at 18000 × g 4℃ for 15 min, the supernatant was removed and filtered through 0.22μm membrane. For LC/MS based metabolomics analysis, the dried samples were dissolved in 200μL water and 5μL aliquots were analyzed.

Combined analysis:

Analysis ID	AN003483	AN003484
Analysis type	MS	MS
Chromatography type	Reversed phase	Reversed phase
Chromatography system	Agilent 1290 Infinity	Agilent 1290 Infinity
Column	Waters Acquity BEH HSS T3 (100 x 2.1mm,1.8um)	Waters Acquity BEH HSS T3 (100 x 2.1mm,1.8um)
MS Type	ESI	ESI
MS instrument type	Triple quadrupole	Triple quadrupole
MS instrument name	Agilent 6495 QQQ	Agilent 6495 QQQ
Ion Mode	POSITIVE	NEGATIVE
Units	peak area	peak area

Chromatography:

Chromatography ID:	CH002572
Instrument Name:	Agilent 1290 Infinity
Column Name:	Waters Acquity BEH HSS T3 (100 x 2.1mm,1.8um)
Chromatography Type:	Reversed phase

MS:

MS ID:	MS003244
Analysis ID:	AN003483
Instrument Name:	Agilent 6495 QQQ
Instrument Type:	Triple quadrupole
MS Type:	ESI
MS Comments:	Agilent MassHunter Workstation Data Acquisition Agilent MassHunter QualitativeAnalysis B.07.00 Agilent MassHunter Quantitative Analysis (for QQQ)
Ion Mode:	POSITIVE

MS ID:	MS003245
Analysis ID:	AN003484
Instrument Name:	Agilent 6495 QQQ
Instrument Type:	Triple quadrupole
MS Type:	ESI
MS Comments:	Agilent MassHunter Workstation Data Acquisition Agilent MassHunter QualitativeAnalysis B.07.00 Agilent MassHunter Quantitative Analysis (for QQQ)
Ion Mode:	NEGATIVE