#METABOLOMICS WORKBENCH Lu_Group_20220325_023225 DATATRACK_ID:3138 STUDY_ID:ST002129 ANALYSIS_ID:AN003482 PROJECT_ID:PR001349 VERSION 1 CREATED_ON April 6, 2022, 7:01 pm #PROJECT PR:PROJECT_TITLE Discovery and characterization of virulence associated functional metabolites in PR:PROJECT_TITLE Escherichia coli based on functional metabolomics strategy PR:PROJECT_TYPE Untargeted MS quantitative analysis PR:PROJECT_SUMMARY Discovery and characterization of virulence associated functional metabolites in PR:PROJECT_SUMMARY Escherichia coli based on functional metabolomics strategy PR:INSTITUTE Shanghai Center for Systems Biomedicine, Shanghai Jiaotong University PR:LAST_NAME Lu PR:FIRST_NAME Haitao PR:ADDRESS 800 Dongchuan RD. Minhang District, Shanghai, Shanghai, 200240, China PR:EMAIL haitao.lu@sjtu.edu.cn PR:PHONE 15221478139 #STUDY ST:STUDY_TITLE Discovery and characterization of virulence associated functional metabolites in ST:STUDY_TITLE Escherichia coli based on functional metabolomics strategy(siderophores ST:STUDY_TITLE metabolomics-2) ST:STUDY_SUMMARY Bacterial metabolites are substrates of virulence factors of uropathogenic ST:STUDY_SUMMARY Escherichia coli (UPEC), but the mechanism underlying the role of functional ST:STUDY_SUMMARY metabolites in bacterial virulence from the perspective of small molecular ST:STUDY_SUMMARY metabolism is unclear. In the present study, we used a strategy of functional ST:STUDY_SUMMARY metabolomics integrated with bacterial genetics in attempt to decipher the ST:STUDY_SUMMARY mechanism of virulence formation in Escherichia coli (E. coli) from the ST:STUDY_SUMMARY viewpoint of small molecule metabolism. We identified the virulence-associated ST:STUDY_SUMMARY metabolome via analysis of the primary metabolome of the pathogenic UTI89 strain ST:STUDY_SUMMARY and the non-pathogenic MG1655 strain. Then, the iron-mediated virulence ST:STUDY_SUMMARY associated metabolome was identified by an iron fishing strategy. Also, the ST:STUDY_SUMMARY mechanism of siderophores in regulating pathogenicity in different environments ST:STUDY_SUMMARY was explored by investigating the effect of iron on siderophore biosynthesis. ST:STUDY_SUMMARY Finally, by knocking out genes related to siderophore biosynthesis, siderophore ST:STUDY_SUMMARY transport and iron utilization, siderophores dependent iron-regulating virulence ST:STUDY_SUMMARY associated metabolome, including 18 functional metabolites, was identified and ST:STUDY_SUMMARY verified to be involved in the regulation of bacterial virulence. Based on this ST:STUDY_SUMMARY we found that these functional metabolites regulated the virulence of E. coli by ST:STUDY_SUMMARY targeting multiple metabolic pathways in an iron-siderophores dependent manner. ST:STUDY_SUMMARY Moreover, a quantitative proteomics approach was implemented to further ST:STUDY_SUMMARY elucidate the mechanism of functional metabolites and functional proteins in ST:STUDY_SUMMARY modulating bacterial virulence. And our findings demonstrated that functional ST:STUDY_SUMMARY proteins regulated the virulence of E. coli by mediating iron binding, ST:STUDY_SUMMARY iron-siderophore transmembrane transport, and the biosynthesis and expression of ST:STUDY_SUMMARY functional metabolites. Interestingly, we found that functional metabolites ST:STUDY_SUMMARY enhance the virulence of E. coli by specifically modulating the key metabolic ST:STUDY_SUMMARY pathways involved in purine metabolism, proline metabolism, arginine metabolism ST:STUDY_SUMMARY and pyrimidine metabolism. Taken together, our study identified for the first ST:STUDY_SUMMARY time 18 functional metabolites regulating the of E. coli virulence, greatly ST:STUDY_SUMMARY enriching our understanding of the mechanism of functional metabolites that ST:STUDY_SUMMARY regulate the E. coli virulence by targeting primary metabolism, which will ST:STUDY_SUMMARY largely contribute to the development of new strategies to target ST:STUDY_SUMMARY virulence-based diagnosis and therapy of infections caused by different ST:STUDY_SUMMARY pathogens. ST:INSTITUTE Shanghai Center for Systems Biomedicine, Shanghai Jiaotong University ST:LAST_NAME Lu ST:FIRST_NAME Haitao ST:ADDRESS 800 Dongchuan RD. Minhang District, Shanghai, Shanghai, 200240, China ST:EMAIL haitao.lu@sjtu.edu.cn ST:PHONE 15221478139 #SUBJECT SU:SUBJECT_TYPE Bacteria SU:SUBJECT_SPECIES Escherichia coli SU:TAXONOMY_ID 562 #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data SUBJECT_SAMPLE_FACTORS - WT-0-1S Treatment:standard growth conditions RAW_FILE_NAME=WT-0-1S SUBJECT_SAMPLE_FACTORS - WT-0-2S Treatment:standard growth conditions RAW_FILE_NAME=WT-0-2S SUBJECT_SAMPLE_FACTORS - WT-0-3S Treatment:standard growth conditions RAW_FILE_NAME=WT-0-3S SUBJECT_SAMPLE_FACTORS - WT-0-4S Treatment:standard growth conditions RAW_FILE_NAME=WT-0-4S SUBJECT_SAMPLE_FACTORS - WT-0-5S Treatment:standard growth conditions RAW_FILE_NAME=WT-0-5S SUBJECT_SAMPLE_FACTORS - WT-0-6S Treatment:standard growth conditions RAW_FILE_NAME=WT-0-6S SUBJECT_SAMPLE_FACTORS - WT-10-1S Treatment:10 μM iron supplementation RAW_FILE_NAME=WT-10-1S SUBJECT_SAMPLE_FACTORS - WT-10-2S Treatment:10 μM iron supplementation RAW_FILE_NAME=WT-10-2S SUBJECT_SAMPLE_FACTORS - WT-10-3S Treatment:10 μM iron supplementation RAW_FILE_NAME=WT-10-3S SUBJECT_SAMPLE_FACTORS - WT-10-4S Treatment:10 μM iron supplementation RAW_FILE_NAME=WT-10-4S SUBJECT_SAMPLE_FACTORS - WT-10-5S Treatment:10 μM iron supplementation RAW_FILE_NAME=WT-10-5S SUBJECT_SAMPLE_FACTORS - WT-10-6S Treatment:10 μM iron supplementation RAW_FILE_NAME=WT-10-6S SUBJECT_SAMPLE_FACTORS - MG1655-0-1S Treatment:standard growth conditions RAW_FILE_NAME=MG1655-0-1S SUBJECT_SAMPLE_FACTORS - MG1655-0-2S Treatment:standard growth conditions RAW_FILE_NAME=MG1655-0-2S SUBJECT_SAMPLE_FACTORS - MG1655-0-3S Treatment:standard growth conditions RAW_FILE_NAME=MG1655-0-3S SUBJECT_SAMPLE_FACTORS - MG1655-0-4S Treatment:standard growth conditions RAW_FILE_NAME=MG1655-0-4S SUBJECT_SAMPLE_FACTORS - MG1655-0-5S Treatment:standard growth conditions RAW_FILE_NAME=MG1655-0-5S SUBJECT_SAMPLE_FACTORS - MG1655-0-6S Treatment:standard growth conditions RAW_FILE_NAME=MG1655-0-6S SUBJECT_SAMPLE_FACTORS - MG1655-10-1S Treatment:10 μM iron supplementation RAW_FILE_NAME=MG1655-10-1S SUBJECT_SAMPLE_FACTORS - MG1655-10-2S Treatment:10 μM iron supplementation RAW_FILE_NAME=MG1655-10-2S SUBJECT_SAMPLE_FACTORS - MG1655-10-3S Treatment:10 μM iron supplementation RAW_FILE_NAME=MG1655-10-3S SUBJECT_SAMPLE_FACTORS - MG1655-10-4S Treatment:10 μM iron supplementation RAW_FILE_NAME=MG1655-10-4S SUBJECT_SAMPLE_FACTORS - MG1655-10-5S Treatment:10 μM iron supplementation RAW_FILE_NAME=MG1655-10-5S SUBJECT_SAMPLE_FACTORS - MG1655-10-6S Treatment:10 μM iron supplementation RAW_FILE_NAME=MG1655-10-6S #COLLECTION CO:COLLECTION_SUMMARY After 18h of culture, the sample supernatant was isolated.Then, 2μL 0.1M ferric CO:COLLECTION_SUMMARY chloride was mixed with 2 mL of cell supernatant. After incubating at room CO:COLLECTION_SUMMARY temperature for 15 minutes, the precipitate was removed by centrifugation at CO:COLLECTION_SUMMARY 20000 × g for 15 min at 4 °C. The supernatant was added to an SPE plate CO:COLLECTION_SUMMARY (Waters, Oasis HLB) and washed with 0.5 mL 5% methanol, and then eluted with 0.5 CO:COLLECTION_SUMMARY mL 100% methanol to obtain the siderophores. CO:SAMPLE_TYPE Bacterial cells #TREATMENT TR:TREATMENT_SUMMARY M63 medium (1.36% monopotassium phosphate, 0.2% ammonium sulfate, 0.024% TR:TREATMENT_SUMMARY magnesium sulfate, 0.001% calcium chloride, and 0.0015% nicotinic acid) was used TR:TREATMENT_SUMMARY to form MG1655 and UTI89. In addition, add ferric chloride solution to the TR:TREATMENT_SUMMARY medium to prepare 10μM iron M63 medium, we cultured the wild UTI89 strain and TR:TREATMENT_SUMMARY MG1655 in the presence of 10μM iron. The E. coli strain was incubated in TR:TREATMENT_SUMMARY LB-agar plate for 12 hours, one colony was isolated to LB broth for further 4 TR:TREATMENT_SUMMARY hours incubation, then diluted the solution into M63 medium at a ratio of 1:100 TR:TREATMENT_SUMMARY and the cultures were incubated for another18 h at 37°C, 200rpm to culture E. TR:TREATMENT_SUMMARY coli. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Siderophores were extracted as previously described. Briefly, 12μL 0.1M ferric SP:SAMPLEPREP_SUMMARY chloride was mixed with 2 mL of cell supernatant. After incubating at room SP:SAMPLEPREP_SUMMARY temperature for 15 minutes, the precipitate was removed by centrifugation at SP:SAMPLEPREP_SUMMARY 20000 × g for 15 min at 4 °C. The supernatant was added to an SPE plate SP:SAMPLEPREP_SUMMARY (Waters, Oasis HLB) and washed with 0.5 mL 5% methanol, and then eluted with 0.5 SP:SAMPLEPREP_SUMMARY mL 100% methanol to obtain the siderophores. LC/MS analysis was performed using SP:SAMPLEPREP_SUMMARY 5μL aliquots. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Agilent 1290 Infinity CH:COLUMN_NAME Waters Acquity BEH HSS T3 (100 x 2.1mm, 1.8um) #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Agilent 6560 Ion Mobility MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS Agilent MassHunter Workstation Data Acquisition Agilent MassHunter MS:MS_COMMENTS QualitativeAnalysis B.07.00 Agilent MassHunter Quantitative Analysis (for QTOF) #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS peak area MS_METABOLITE_DATA_START Samples WT-0-1S WT-0-2S WT-0-3S WT-0-4S WT-0-5S WT-0-6S WT-10-1S WT-10-2S WT-10-3S WT-10-4S WT-10-5S WT-10-6S MG1655-0-1S MG1655-0-2S MG1655-0-3S MG1655-0-4S MG1655-0-5S MG1655-0-6S MG1655-10-1S MG1655-10-2S MG1655-10-3S MG1655-10-4S MG1655-10-5S MG1655-10-6S Factors Treatment:standard growth conditions Treatment:standard growth conditions Treatment:standard growth conditions Treatment:standard growth conditions Treatment:standard growth conditions Treatment:standard growth conditions Treatment:10 μM iron supplementation Treatment:10 μM iron supplementation Treatment:10 μM iron supplementation Treatment:10 μM iron supplementation Treatment:10 μM iron supplementation Treatment:10 μM iron supplementation Treatment:standard growth conditions Treatment:standard growth conditions Treatment:standard growth conditions Treatment:standard growth conditions Treatment:standard growth conditions Treatment:standard growth conditions Treatment:10 μM iron supplementation Treatment:10 μM iron supplementation Treatment:10 μM iron supplementation Treatment:10 μM iron supplementation Treatment:10 μM iron supplementation Treatment:10 μM iron supplementation enterobactin 515292 615239 1060330 811184 419824 466272 336343 321808 667505 572655 476851 349078 3925844 3562670 4083195 4843017 658000 5376059 727156 741385 678088 522021 621916 397058 enterobactin-Fe 62142 62230 72926 23830 17410 12823 69089 69268 69453 70481 46937 59166 57347 56823 46856 41172 47040 48853 129809 124791 106956 67245 56843 107872 yersiniabactin 24577148 10157895 18141733 19752677 15728024 14104995 42776243 43629630 28450665 38267643 41417168 22122513 yersiniabactin+Fe 97791390 49671046 80670582 103884135 82423069 75897153 24593491 32865551 53690948 41526008 51420622 38557891 HPTT 58461939 36684314 40962049 67832705 46430761 36272247 27777502 29098864 28758707 26629458 31125663 19824542 HPTT-Fe 438625 139389 256774 500994 518944 392365 1331638 1520403 1651667 1717536 2044592 1288922 salmochelin 142466 83882 134012 285077 75737 86470 60950 23520 49702 35707 35307 48976 salmochelin-Fe 22428 23574 26365 17916 24707 17744 28496 32494 29713 30327 33146 30975 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name quantified m/z retention time enterobactin 670.1652 16.42 enterobactin-Fe 723.0616 16.42 yersiniabactin 482.1342 17.92 HPTT 307.0206 18.22 salmochelin 404.1182 19.24 salmochelin-Fe 457.2826 19.41 yersiniabactin+Fe 535.0356 15.4 HPTT-Fe 665.9588 18.12 METABOLITES_END #END