#METABOLOMICS WORKBENCH Lu_Group_20220325_061043 DATATRACK_ID:3140 STUDY_ID:ST002130 ANALYSIS_ID:AN003484 PROJECT_ID:PR001349 VERSION 1 CREATED_ON April 6, 2022, 7:17 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_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（pellets ST:STUDY_TITLE metabolomics-1） 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-0-1P Treatment:standard growth conditions RAW_FILE_NAME=WT-0-1P SUBJECT_SAMPLE_FACTORS - WT-0-2P Treatment:standard growth conditions RAW_FILE_NAME=WT-0-2P SUBJECT_SAMPLE_FACTORS - WT-0-3P Treatment:standard growth conditions RAW_FILE_NAME=WT-0-3P SUBJECT_SAMPLE_FACTORS - WT-0-4P Treatment:standard growth conditions RAW_FILE_NAME=WT-0-4P SUBJECT_SAMPLE_FACTORS - WT-0-5P Treatment:standard growth conditions RAW_FILE_NAME=WT-0-5P SUBJECT_SAMPLE_FACTORS - WT-0-6P Treatment:standard growth conditions RAW_FILE_NAME=WT-0-6P SUBJECT_SAMPLE_FACTORS - WT-10-1P Treatment:10 μM iron supplementation RAW_FILE_NAME=WT-10-1P SUBJECT_SAMPLE_FACTORS - WT-10-2P Treatment:10 μM iron supplementation RAW_FILE_NAME=WT-10-2P SUBJECT_SAMPLE_FACTORS - WT-10-3P Treatment:10 μM iron supplementation RAW_FILE_NAME=WT-10-3P SUBJECT_SAMPLE_FACTORS - WT-10-4P Treatment:10 μM iron supplementation RAW_FILE_NAME=WT-10-4P SUBJECT_SAMPLE_FACTORS - WT-10-5P Treatment:10 μM iron supplementation RAW_FILE_NAME=WT-10-5P SUBJECT_SAMPLE_FACTORS - WT-10-6P Treatment:10 μM iron supplementation RAW_FILE_NAME=WT-10-6P SUBJECT_SAMPLE_FACTORS - MG1655-0-1P Treatment:standard growth conditions RAW_FILE_NAME=MG1655-0-1P SUBJECT_SAMPLE_FACTORS - MG1655-0-2P Treatment:standard growth conditions RAW_FILE_NAME=MG1655-0-2P SUBJECT_SAMPLE_FACTORS - MG1655-0-3P Treatment:standard growth conditions RAW_FILE_NAME=MG1655-0-3P SUBJECT_SAMPLE_FACTORS - MG1655-0-4P Treatment:standard growth conditions RAW_FILE_NAME=MG1655-0-4P SUBJECT_SAMPLE_FACTORS - MG1655-0-5P Treatment:standard growth conditions RAW_FILE_NAME=MG1655-0-5P SUBJECT_SAMPLE_FACTORS - MG1655-0-6P Treatment:standard growth conditions RAW_FILE_NAME=MG1655-0-6P SUBJECT_SAMPLE_FACTORS - ybtS-0-1P Treatment:standard growth conditions RAW_FILE_NAME=ybtS-0-1P SUBJECT_SAMPLE_FACTORS - ybtS-0-2P Treatment:standard growth conditions RAW_FILE_NAME=ybtS-0-2P SUBJECT_SAMPLE_FACTORS - ybtS-0-3P Treatment:standard growth conditions RAW_FILE_NAME=ybtS-0-3P SUBJECT_SAMPLE_FACTORS - ybtS-0-4P Treatment:standard growth conditions RAW_FILE_NAME=ybtS-0-4P SUBJECT_SAMPLE_FACTORS - ybtS-0-5P Treatment:standard growth conditions RAW_FILE_NAME=ybtS-0-5P SUBJECT_SAMPLE_FACTORS - ybtS-0-6P Treatment:standard growth conditions RAW_FILE_NAME=ybtS-0-6P SUBJECT_SAMPLE_FACTORS - BS-0-1P Treatment:standard growth conditions RAW_FILE_NAME=BS-0-1P SUBJECT_SAMPLE_FACTORS - BS-0-2P Treatment:standard growth conditions RAW_FILE_NAME=BS-0-2P SUBJECT_SAMPLE_FACTORS - BS-0-3P Treatment:standard growth conditions RAW_FILE_NAME=BS-0-3P SUBJECT_SAMPLE_FACTORS - BS-0-4P Treatment:standard growth conditions RAW_FILE_NAME=BS-0-4P SUBJECT_SAMPLE_FACTORS - BS-0-5P Treatment:standard growth conditions RAW_FILE_NAME=BS-0-5P SUBJECT_SAMPLE_FACTORS - BS-0-6P Treatment:standard growth conditions RAW_FILE_NAME=BS-0-6P SUBJECT_SAMPLE_FACTORS - entB-0-1P Treatment:standard growth conditions RAW_FILE_NAME=entB-0-1P SUBJECT_SAMPLE_FACTORS - entB-0-2P Treatment:standard growth conditions RAW_FILE_NAME=entB-0-2P SUBJECT_SAMPLE_FACTORS - entB-0-3P Treatment:standard growth conditions RAW_FILE_NAME=entB-0-3P SUBJECT_SAMPLE_FACTORS - entB-0-4P Treatment:standard growth conditions RAW_FILE_NAME=entB-0-4P SUBJECT_SAMPLE_FACTORS - entB-0-5P Treatment:standard growth conditions RAW_FILE_NAME=entB-0-5P SUBJECT_SAMPLE_FACTORS - entB-0-6P Treatment:standard growth conditions RAW_FILE_NAME=entB-0-6P SUBJECT_SAMPLE_FACTORS - iroA-0-1P Treatment:standard growth conditions RAW_FILE_NAME=iroA-0-1P SUBJECT_SAMPLE_FACTORS - iroA-0-2P Treatment:standard growth conditions RAW_FILE_NAME=iroA-0-2P SUBJECT_SAMPLE_FACTORS - iroA-0-3P Treatment:standard growth conditions RAW_FILE_NAME=iroA-0-3P SUBJECT_SAMPLE_FACTORS - iroA-0-4P Treatment:standard growth conditions RAW_FILE_NAME=iroA-0-4P SUBJECT_SAMPLE_FACTORS - iroA-0-5P Treatment:standard growth conditions RAW_FILE_NAME=iroA-0-5P SUBJECT_SAMPLE_FACTORS - iroA-0-6P Treatment:standard growth conditions RAW_FILE_NAME=iroA-0-6P SUBJECT_SAMPLE_FACTORS - irp1-0-1P Treatment:standard growth conditions RAW_FILE_NAME=irp1-0-1P SUBJECT_SAMPLE_FACTORS - irp1-0-2P Treatment:standard growth conditions RAW_FILE_NAME=irp1-0-2P SUBJECT_SAMPLE_FACTORS - irp1-0-3P Treatment:standard growth conditions RAW_FILE_NAME=irp1-0-3P SUBJECT_SAMPLE_FACTORS - irp1-0-4P Treatment:standard growth conditions RAW_FILE_NAME=irp1-0-4P SUBJECT_SAMPLE_FACTORS - irp1-0-5P Treatment:standard growth conditions RAW_FILE_NAME=irp1-0-5P SUBJECT_SAMPLE_FACTORS - irp1-0-6P Treatment:standard growth conditions RAW_FILE_NAME=irp1-0-6P #COLLECTION CO:COLLECTION_SUMMARY After 18h of culture, the sample pellet was isolated. The bacterial pellets CO:COLLECTION_SUMMARY harvested from 50 mL of broth culture were mixed with 1.2 mL 80% ice-cold CO:COLLECTION_SUMMARY methanol (added to internal standard 0.001mg/ml 4-chloro-DL-phenylalanine), then CO:COLLECTION_SUMMARY vortexed for 30 s, and placed on dry ice for 30 min. The samples were CO:COLLECTION_SUMMARY centrifuged at 18000 × g for 15 min at 4 °C. The frozen samples were ground CO:COLLECTION_SUMMARY with beads and the homogenates were centrifuged at 18000 × g for 15 min at 4 CO:COLLECTION_SUMMARY °C. The supernatant was mixed with 800μL ice-cold acetonitrile, and then left CO:COLLECTION_SUMMARY to stand for 20 minutes in an ice bath. After centrifugation at 18000 × g 4℃ CO:COLLECTION_SUMMARY for 15 min, the supernatant was removed and filtered through 0.22μm membrane. 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 mutants. In addition, add ferric chloride solution to TR:TREATMENT_SUMMARY the medium to prepare 10μM iron M63 medium, we cultured the wild UTI89 strain TR:TREATMENT_SUMMARY in the presence of 10μM iron. The E. coli strain was incubated in LB-agar plate TR:TREATMENT_SUMMARY for 12 hours, one colony was isolated to LB broth for further 4 hours TR:TREATMENT_SUMMARY incubation, then diluted the solution into M63 medium at a ratio of 1:100 and TR:TREATMENT_SUMMARY the cultures were incubated for another18 h at 37°C, 200rpm to culture E. coli. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY The bacterial pellets harvested from 50 mL of broth culture were mixed with 1.2 SP:SAMPLEPREP_SUMMARY mL 80% ice-cold methanol (added to internal standard 0.001mg/ml SP:SAMPLEPREP_SUMMARY 4-chloro-DL-phenylalanine), then vortexed for 30 s, and placed on dry ice for 30 SP:SAMPLEPREP_SUMMARY min. The samples were centrifuged at 18000 × g for 15 min at 4 °C. The frozen SP:SAMPLEPREP_SUMMARY samples were ground with beads and the homogenates were centrifuged at 18000 × SP:SAMPLEPREP_SUMMARY g for 15 min at 4 °C. The supernatant was mixed with 800μL ice-cold SP:SAMPLEPREP_SUMMARY acetonitrile, and then left to stand for 20 minutes in an ice bath. After SP:SAMPLEPREP_SUMMARY centrifugation at 18000 × g 4℃ for 15 min, the supernatant was removed and SP:SAMPLEPREP_SUMMARY filtered through 0.22μm membrane. For LC/MS based metabolomics analysis, the SP:SAMPLEPREP_SUMMARY dried samples were dissolved in 200μL water and 5μL aliquots were analyzed. #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 6495 QQQ MS:INSTRUMENT_TYPE Triple quadrupole MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:MS_COMMENTS Agilent MassHunter Workstation Data Acquisition Agilent MassHunter MS:MS_COMMENTS QualitativeAnalysis B.07.00 Agilent MassHunter Quantitative Analysis (for QQQ) #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS peak area MS_METABOLITE_DATA_START Samples MG1655-0-1P MG1655-0-2P MG1655-0-3P MG1655-0-4P MG1655-0-5P MG1655-0-6P WT-10-1P WT-10-2P WT-10-3P WT-10-4P WT-10-5P WT-10-6P WT-0-1P WT-0-2P WT-0-3P WT-0-4P WT-0-5P WT-0-6P entB-0-1P entB-0-2P entB-0-3P entB-0-4P entB-0-5P entB-0-6P BS-0-1P BS-0-2P BS-0-3P BS-0-4P BS-0-5P BS-0-6P iroA-0-1P iroA-0-2P iroA-0-3P iroA-0-4P iroA-0-5P iroA-0-6P irp1-0-1P irp1-0-2P irp1-0-3P irp1-0-4P irp1-0-5P irp1-0-6P ybtS-0-1P ybtS-0-2P ybtS-0-3P ybtS-0-4P ybtS-0-5P ybtS-0-6P 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:standard growth conditions 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:standard growth conditions 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:standard growth conditions 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:standard growth conditions 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:standard growth conditions Treatment:standard growth conditions Methylmalonic acid 15886.67533 14979.46953 16128.03831 19540.04755 16856.23819 16883.09491 864.6740949 873.935898 719.7583998 816.8343121 839.4984105 879.8371413 N-Acetylglutamic acid 5894.307325 5161.855381 6700.462988 6868.011254 6397.570483 7511.019404 869.5037864 853.4397538 861.4746545 802.5332573 796.7948883 833.4401285 Taurine 2060.916716 2010.204166 1933.325361 2170.547825 1944.057819 1742.306661 1114.605616 1119.20977 881.8938483 1001.459268 1125.187052 1121.289599 7276.752501 6308.136434 6535.738346 6478.212742 6169.148328 7159.749476 848.8529614 704.2515697 717.4215393 842.0849196 783.4161713 756.2393784 1217.893386 888.0209963 912.0445293 1366.454218 1215.239778 995.5158052 1266.418895 1358.848497 1291.906978 1295.890744 1388.118482 1368.700728 2009.450422 2416.265979 2240.568319 3125.42294 2403.866015 2846.577172 3415.678184 4063.329662 4004.709286 4080.76379 3666.177006 3960.079185 Succinic acid 18341.50987 18536.83868 19025.28018 19771.9967 18306.34377 17389.82842 364.0195446 413.8180616 284.3214529 247.9484219 354.2136957 271.4230512 781.1351289 753.8401569 828.9134793 859.6450979 829.7897251 742.6408176 Maleic acid 11239.81086 10489.16083 12215.52441 13152.15358 11210.19049 11520.87943 115.384283 82.35704228 92.32343829 102.7209452 116.0555979 64.44312808 5265.846938 6095.311298 5153.977666 6228.743294 4914.952111 6953.762017 617.012918 618.4684446 558.4148459 541.9167611 439.9402726 459.3907969 748.3873022 794.4554308 594.7816207 972.0749857 774.1223362 878.9350269 823.5319799 686.7394514 719.8318665 655.6956456 824.3973722 633.2966563 504.5440111 518.6202144 511.877982 617.7852263 522.5309579 496.4372689 472.0794004 416.6031663 408.0818319 422.1937869 395.815552 440.4002042 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name Molecular weight RT (min) m/z Methylmalonic acid 118.09 2.74 117.02 Maleic acid 116.07 1.1 133 N-Acetylglutamic acid 189.17 1.88 188.05/128 Taurine 125.15 0.9 126 Succinic acid 118 2.31 117.02 METABOLITES_END #END