{
"METABOLOMICS WORKBENCH":{"STUDY_ID":"ST002130","ANALYSIS_ID":"AN003483","VERSION":"1","CREATED_ON":"April 6, 2022, 7:17 pm"},

"PROJECT":{"PROJECT_TITLE":"Discovery and characterization of virulence associated functional metabolites in Escherichia coli based on functional metabolomics strategy","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","LAST_NAME":"Lu","FIRST_NAME":"Haitao","ADDRESS":"800 Dongchuan RD. Minhang District, Shanghai, Shanghai, 200240, China","EMAIL":"haitao.lu@sjtu.edu.cn","PHONE":"15221478139"},

"STUDY":{"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"},

"SUBJECT":{"SUBJECT_TYPE":"Bacteria","SUBJECT_SPECIES":"Escherichia coli","TAXONOMY_ID":"562"},
"SUBJECT_SAMPLE_FACTORS":[
{
"Subject ID":"-",
"Sample ID":"WT-0-1P",
"Factors":{"Treatment":"standard growth conditions"},
"Additional sample data":{"RAW_FILE_NAME":"WT-0-1P"}
},
{
"Subject ID":"-",
"Sample ID":"WT-0-2P",
"Factors":{"Treatment":"standard growth conditions"},
"Additional sample data":{"RAW_FILE_NAME":"WT-0-2P"}
},
{
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"Sample ID":"WT-0-3P",
"Factors":{"Treatment":"standard growth conditions"},
"Additional sample data":{"RAW_FILE_NAME":"WT-0-3P"}
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{
"Subject ID":"-",
"Sample ID":"WT-0-4P",
"Factors":{"Treatment":"standard growth conditions"},
"Additional sample data":{"RAW_FILE_NAME":"WT-0-4P"}
},
{
"Subject ID":"-",
"Sample ID":"WT-0-5P",
"Factors":{"Treatment":"standard growth conditions"},
"Additional sample data":{"RAW_FILE_NAME":"WT-0-5P"}
},
{
"Subject ID":"-",
"Sample ID":"WT-0-6P",
"Factors":{"Treatment":"standard growth conditions"},
"Additional sample data":{"RAW_FILE_NAME":"WT-0-6P"}
},
{
"Subject ID":"-",
"Sample ID":"WT-10-1P",
"Factors":{"Treatment":"10 μM iron supplementation"},
"Additional sample data":{"RAW_FILE_NAME":"WT-10-1P"}
},
{
"Subject ID":"-",
"Sample ID":"WT-10-2P",
"Factors":{"Treatment":"10 μM iron supplementation"},
"Additional sample data":{"RAW_FILE_NAME":"WT-10-2P"}
},
{
"Subject ID":"-",
"Sample ID":"WT-10-3P",
"Factors":{"Treatment":"10 μM iron supplementation"},
"Additional sample data":{"RAW_FILE_NAME":"WT-10-3P"}
},
{
"Subject ID":"-",
"Sample ID":"WT-10-4P",
"Factors":{"Treatment":"10 μM iron supplementation"},
"Additional sample data":{"RAW_FILE_NAME":"WT-10-4P"}
},
{
"Subject ID":"-",
"Sample ID":"WT-10-5P",
"Factors":{"Treatment":"10 μM iron supplementation"},
"Additional sample data":{"RAW_FILE_NAME":"WT-10-5P"}
},
{
"Subject ID":"-",
"Sample ID":"WT-10-6P",
"Factors":{"Treatment":"10 μM iron supplementation"},
"Additional sample data":{"RAW_FILE_NAME":"WT-10-6P"}
},
{
"Subject ID":"-",
"Sample ID":"MG1655-0-1P",
"Factors":{"Treatment":"standard growth conditions"},
"Additional sample data":{"RAW_FILE_NAME":"MG1655-0-1P"}
},
{
"Subject ID":"-",
"Sample ID":"MG1655-0-2P",
"Factors":{"Treatment":"standard growth conditions"},
"Additional sample data":{"RAW_FILE_NAME":"MG1655-0-2P"}
},
{
"Subject ID":"-",
"Sample ID":"MG1655-0-3P",
"Factors":{"Treatment":"standard growth conditions"},
"Additional sample data":{"RAW_FILE_NAME":"MG1655-0-3P"}
},
{
"Subject ID":"-",
"Sample ID":"MG1655-0-4P",
"Factors":{"Treatment":"standard growth conditions"},
"Additional sample data":{"RAW_FILE_NAME":"MG1655-0-4P"}
},
{
"Subject ID":"-",
"Sample ID":"MG1655-0-5P",
"Factors":{"Treatment":"standard growth conditions"},
"Additional sample data":{"RAW_FILE_NAME":"MG1655-0-5P"}
},
{
"Subject ID":"-",
"Sample ID":"MG1655-0-6P",
"Factors":{"Treatment":"standard growth conditions"},
"Additional sample data":{"RAW_FILE_NAME":"MG1655-0-6P"}
},
{
"Subject ID":"-",
"Sample ID":"ybtS-0-1P",
"Factors":{"Treatment":"standard growth conditions"},
"Additional sample data":{"RAW_FILE_NAME":"ybtS-0-1P"}
},
{
"Subject ID":"-",
"Sample ID":"ybtS-0-2P",
"Factors":{"Treatment":"standard growth conditions"},
"Additional sample data":{"RAW_FILE_NAME":"ybtS-0-2P"}
},
{
"Subject ID":"-",
"Sample ID":"ybtS-0-3P",
"Factors":{"Treatment":"standard growth conditions"},
"Additional sample data":{"RAW_FILE_NAME":"ybtS-0-3P"}
},
{
"Subject ID":"-",
"Sample ID":"ybtS-0-4P",
"Factors":{"Treatment":"standard growth conditions"},
"Additional sample data":{"RAW_FILE_NAME":"ybtS-0-4P"}
},
{
"Subject ID":"-",
"Sample ID":"ybtS-0-5P",
"Factors":{"Treatment":"standard growth conditions"},
"Additional sample data":{"RAW_FILE_NAME":"ybtS-0-5P"}
},
{
"Subject ID":"-",
"Sample ID":"ybtS-0-6P",
"Factors":{"Treatment":"standard growth conditions"},
"Additional sample data":{"RAW_FILE_NAME":"ybtS-0-6P"}
},
{
"Subject ID":"-",
"Sample ID":"BS-0-1P",
"Factors":{"Treatment":"standard growth conditions"},
"Additional sample data":{"RAW_FILE_NAME":"BS-0-1P"}
},
{
"Subject ID":"-",
"Sample ID":"BS-0-2P",
"Factors":{"Treatment":"standard growth conditions"},
"Additional sample data":{"RAW_FILE_NAME":"BS-0-2P"}
},
{
"Subject ID":"-",
"Sample ID":"BS-0-3P",
"Factors":{"Treatment":"standard growth conditions"},
"Additional sample data":{"RAW_FILE_NAME":"BS-0-3P"}
},
{
"Subject ID":"-",
"Sample ID":"BS-0-4P",
"Factors":{"Treatment":"standard growth conditions"},
"Additional sample data":{"RAW_FILE_NAME":"BS-0-4P"}
},
{
"Subject ID":"-",
"Sample ID":"BS-0-5P",
"Factors":{"Treatment":"standard growth conditions"},
"Additional sample data":{"RAW_FILE_NAME":"BS-0-5P"}
},
{
"Subject ID":"-",
"Sample ID":"BS-0-6P",
"Factors":{"Treatment":"standard growth conditions"},
"Additional sample data":{"RAW_FILE_NAME":"BS-0-6P"}
},
{
"Subject ID":"-",
"Sample ID":"entB-0-1P",
"Factors":{"Treatment":"standard growth conditions"},
"Additional sample data":{"RAW_FILE_NAME":"entB-0-1P"}
},
{
"Subject ID":"-",
"Sample ID":"entB-0-2P",
"Factors":{"Treatment":"standard growth conditions"},
"Additional sample data":{"RAW_FILE_NAME":"entB-0-2P"}
},
{
"Subject ID":"-",
"Sample ID":"entB-0-3P",
"Factors":{"Treatment":"standard growth conditions"},
"Additional sample data":{"RAW_FILE_NAME":"entB-0-3P"}
},
{
"Subject ID":"-",
"Sample ID":"entB-0-4P",
"Factors":{"Treatment":"standard growth conditions"},
"Additional sample data":{"RAW_FILE_NAME":"entB-0-4P"}
},
{
"Subject ID":"-",
"Sample ID":"entB-0-5P",
"Factors":{"Treatment":"standard growth conditions"},
"Additional sample data":{"RAW_FILE_NAME":"entB-0-5P"}
},
{
"Subject ID":"-",
"Sample ID":"entB-0-6P",
"Factors":{"Treatment":"standard growth conditions"},
"Additional sample data":{"RAW_FILE_NAME":"entB-0-6P"}
},
{
"Subject ID":"-",
"Sample ID":"iroA-0-1P",
"Factors":{"Treatment":"standard growth conditions"},
"Additional sample data":{"RAW_FILE_NAME":"iroA-0-1P"}
},
{
"Subject ID":"-",
"Sample ID":"iroA-0-2P",
"Factors":{"Treatment":"standard growth conditions"},
"Additional sample data":{"RAW_FILE_NAME":"iroA-0-2P"}
},
{
"Subject ID":"-",
"Sample ID":"iroA-0-3P",
"Factors":{"Treatment":"standard growth conditions"},
"Additional sample data":{"RAW_FILE_NAME":"iroA-0-3P"}
},
{
"Subject ID":"-",
"Sample ID":"iroA-0-4P",
"Factors":{"Treatment":"standard growth conditions"},
"Additional sample data":{"RAW_FILE_NAME":"iroA-0-4P"}
},
{
"Subject ID":"-",
"Sample ID":"iroA-0-5P",
"Factors":{"Treatment":"standard growth conditions"},
"Additional sample data":{"RAW_FILE_NAME":"iroA-0-5P"}
},
{
"Subject ID":"-",
"Sample ID":"iroA-0-6P",
"Factors":{"Treatment":"standard growth conditions"},
"Additional sample data":{"RAW_FILE_NAME":"iroA-0-6P"}
},
{
"Subject ID":"-",
"Sample ID":"irp1-0-1P",
"Factors":{"Treatment":"standard growth conditions"},
"Additional sample data":{"RAW_FILE_NAME":"irp1-0-1P"}
},
{
"Subject ID":"-",
"Sample ID":"irp1-0-2P",
"Factors":{"Treatment":"standard growth conditions"},
"Additional sample data":{"RAW_FILE_NAME":"irp1-0-2P"}
},
{
"Subject ID":"-",
"Sample ID":"irp1-0-3P",
"Factors":{"Treatment":"standard growth conditions"},
"Additional sample data":{"RAW_FILE_NAME":"irp1-0-3P"}
},
{
"Subject ID":"-",
"Sample ID":"irp1-0-4P",
"Factors":{"Treatment":"standard growth conditions"},
"Additional sample data":{"RAW_FILE_NAME":"irp1-0-4P"}
},
{
"Subject ID":"-",
"Sample ID":"irp1-0-5P",
"Factors":{"Treatment":"standard growth conditions"},
"Additional sample data":{"RAW_FILE_NAME":"irp1-0-5P"}
},
{
"Subject ID":"-",
"Sample ID":"irp1-0-6P",
"Factors":{"Treatment":"standard growth conditions"},
"Additional sample data":{"RAW_FILE_NAME":"irp1-0-6P"}
}
],
"COLLECTION":{"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_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."},

"SAMPLEPREP":{"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."},

"CHROMATOGRAPHY":{"CHROMATOGRAPHY_TYPE":"Reversed phase","INSTRUMENT_NAME":"Agilent 1290 Infinity","COLUMN_NAME":"Waters Acquity BEH HSS T3 (100 x 2.1mm, 1.8um)"},

"ANALYSIS":{"ANALYSIS_TYPE":"MS"},

"MS":{"INSTRUMENT_NAME":"Agilent 6495 QQQ","INSTRUMENT_TYPE":"Triple quadrupole","MS_TYPE":"ESI","ION_MODE":"POSITIVE","MS_COMMENTS":"Agilent MassHunter Workstation Data Acquisition Agilent MassHunter QualitativeAnalysis B.07.00 Agilent MassHunter Quantitative Analysis (for QQQ)"},

"MS_METABOLITE_DATA":{
"Units":"peak area",

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}

}