#METABOLOMICS WORKBENCH Lu_Group_20220325_181827 DATATRACK_ID:3141 STUDY_ID:ST002131 ANALYSIS_ID:AN003486 PROJECT_ID:PR001349 VERSION 1 CREATED_ON April 6, 2022, 7:20 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-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 - ybtP-0-1P Treatment:standard growth conditions RAW_FILE_NAME=ybtP-0-1P SUBJECT_SAMPLE_FACTORS - ybtP-0-2P Treatment:standard growth conditions RAW_FILE_NAME=ybtP-0-2P SUBJECT_SAMPLE_FACTORS - ybtP-0-3P Treatment:standard growth conditions RAW_FILE_NAME=ybtP-0-3P SUBJECT_SAMPLE_FACTORS - ybtP-0-4P Treatment:standard growth conditions RAW_FILE_NAME=ybtP-0-4P SUBJECT_SAMPLE_FACTORS - ybtP-0-5P Treatment:standard growth conditions RAW_FILE_NAME=ybtP-0-5P SUBJECT_SAMPLE_FACTORS - ybtP-0-6P Treatment:standard growth conditions RAW_FILE_NAME=ybtP-0-6P SUBJECT_SAMPLE_FACTORS - ybtQ-0-1P Treatment:standard growth conditions RAW_FILE_NAME=ybtQ-0-1P SUBJECT_SAMPLE_FACTORS - ybtQ-0-2P Treatment:standard growth conditions RAW_FILE_NAME=ybtQ-0-2P SUBJECT_SAMPLE_FACTORS - ybtQ-0-3P Treatment:standard growth conditions RAW_FILE_NAME=ybtQ-0-3P SUBJECT_SAMPLE_FACTORS - ybtQ-0-4P Treatment:standard growth conditions RAW_FILE_NAME=ybtQ-0-4P SUBJECT_SAMPLE_FACTORS - ybtQ-0-5P Treatment:standard growth conditions RAW_FILE_NAME=ybtQ-0-5P SUBJECT_SAMPLE_FACTORS - ybtQ-0-6P Treatment:standard growth conditions RAW_FILE_NAME=ybtQ-0-6P SUBJECT_SAMPLE_FACTORS - fyuA-0-1P Treatment:standard growth conditions RAW_FILE_NAME=fyuA-0-1P SUBJECT_SAMPLE_FACTORS - fyuA-0-2P Treatment:standard growth conditions RAW_FILE_NAME=fyuA-0-2P SUBJECT_SAMPLE_FACTORS - fyuA-0-3P Treatment:standard growth conditions RAW_FILE_NAME=fyuA-0-3P SUBJECT_SAMPLE_FACTORS - fyuA-0-4P Treatment:standard growth conditions RAW_FILE_NAME=fyuA-0-4P SUBJECT_SAMPLE_FACTORS - fyuA-0-5P Treatment:standard growth conditions RAW_FILE_NAME=fyuA-0-5P SUBJECT_SAMPLE_FACTORS - fyuA-0-6P Treatment:standard growth conditions RAW_FILE_NAME=fyuA-0-6P SUBJECT_SAMPLE_FACTORS - fur-0-1P Treatment:No iron supplementation RAW_FILE_NAME=fur-0-1P SUBJECT_SAMPLE_FACTORS - fur-0-2P Treatment:No iron supplementation RAW_FILE_NAME=fur-0-2P SUBJECT_SAMPLE_FACTORS - fur-0-3P Treatment:No iron supplementation RAW_FILE_NAME=fur-0-3P SUBJECT_SAMPLE_FACTORS - fur-0-4P Treatment:No iron supplementation RAW_FILE_NAME=fur-0-4P SUBJECT_SAMPLE_FACTORS - fur-0-5P Treatment:No iron supplementation RAW_FILE_NAME=fur-0-5P SUBJECT_SAMPLE_FACTORS - fur-0-6P Treatment:No iron supplementation RAW_FILE_NAME=fur-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 UTI89 mutants. The E. coli strain was incubated in LB-agar plate for 12 TR:TREATMENT_SUMMARY hours, one colony was isolated to LB broth for further 4 hours incubation, then TR:TREATMENT_SUMMARY diluted the solution into M63 medium at a ratio of 1:100 and the cultures were TR:TREATMENT_SUMMARY 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-DLphenylalanine), 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 fur-0-1P fur-0-2P fur-0-3P fur-0-4P fur-0-5P fur-0-6P fyuA-0-1P fyuA-0-2P fyuA-0-3P fyuA-0-4P fyuA-0-5P fyuA-0-6P ybtP-0-1P ybtP-0-2P ybtP-0-3P ybtP-0-4P ybtP-0-5P ybtP-0-6P ybtQ-0-1P ybtQ-0-2P ybtQ-0-3P ybtQ-0-4P ybtQ-0-5P ybtQ-0-6P Factors Treatment:No iron supplementation Treatment:No iron supplementation Treatment:No iron supplementation Treatment:No iron supplementation Treatment:No iron supplementation Treatment:No 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 Maleic acid 15.9888602 10.6455 22.7941552 17.972349 15.1550762 17.7595815 14.2305403 9.68080604 13.2228563 17.1743368 16.5044588 18.082273 10.1544922 12.5304594 24.5010523 15.7868923 24.1704099 19.9642533 402.785405 564.075294 371.211952 281.825191 382.249361 552.245621 Taurine 436.08945 316.809189 406.636411 533.951569 352.605143 397.951067 297.974509 239.986029 339.137436 303.827054 331.914074 341.430717 472.988246 630.551694 735.504194 555.283967 702.082692 650.610956 486.74169 410.530261 505.113837 469.830055 691.057993 814.682857 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name Molecular weight RT (min) m/z Maleic acid 116.07 1.1 133 Taurine 125.15 0.9 126 METABOLITES_END #END