#METABOLOMICS WORKBENCH Lu_Group_20220323_183941 DATATRACK_ID:3137 STUDY_ID:ST002128 ANALYSIS_ID:AN003481 PROJECT_ID:PR001349 VERSION 1 CREATED_ON April 6, 2022, 7:12 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:DEPARTMENT Shanghai Center for Systems Biomedicine PR:LABORATORY Lu Group PR:LAST_NAME Lu PR:FIRST_NAME Haitao PR:ADDRESS 800 Dongchuan RD. Minhang District, Shanghai, Shanghai, 200240, China PR:EMAIL longlonghu126@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-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 - ybtP-0-1S Treatment:standard growth conditions RAW_FILE_NAME=ybtP-0-1S SUBJECT_SAMPLE_FACTORS - ybtP-0-2S Treatment:standard growth conditions RAW_FILE_NAME=ybtP-0-2S SUBJECT_SAMPLE_FACTORS - ybtP-0-3S Treatment:standard growth conditions RAW_FILE_NAME=ybtP-0-3S SUBJECT_SAMPLE_FACTORS - ybtP-0-4S Treatment:standard growth conditions RAW_FILE_NAME=ybtP-0-4S SUBJECT_SAMPLE_FACTORS - ybtP-0-5S Treatment:standard growth conditions RAW_FILE_NAME=ybtP-0-5S SUBJECT_SAMPLE_FACTORS - ybtP-0-6S Treatment:standard growth conditions RAW_FILE_NAME=ybtP-0-6S SUBJECT_SAMPLE_FACTORS - ybtQ-0-1S Treatment:standard growth conditions RAW_FILE_NAME=ybtQ-0-1S SUBJECT_SAMPLE_FACTORS - ybtQ-0-2S Treatment:standard growth conditions RAW_FILE_NAME=ybtQ-0-2S SUBJECT_SAMPLE_FACTORS - ybtQ-0-3S Treatment:standard growth conditions RAW_FILE_NAME=ybtQ-0-3S SUBJECT_SAMPLE_FACTORS - ybtQ-0-4S Treatment:standard growth conditions RAW_FILE_NAME=ybtQ-0-4S SUBJECT_SAMPLE_FACTORS - ybtQ-0-5S Treatment:standard growth conditions RAW_FILE_NAME=ybtQ-0-5S SUBJECT_SAMPLE_FACTORS - ybtQ-0-6S Treatment:standard growth conditions RAW_FILE_NAME=ybtQ-0-6S SUBJECT_SAMPLE_FACTORS - fyuA-0-1S Treatment:standard growth conditions RAW_FILE_NAME=fyuA-0-1S SUBJECT_SAMPLE_FACTORS - fyuA-0-2S Treatment:standard growth conditions RAW_FILE_NAME=fyuA-0-2S SUBJECT_SAMPLE_FACTORS - fyuA-0-3S Treatment:standard growth conditions RAW_FILE_NAME=fyuA-0-3S SUBJECT_SAMPLE_FACTORS - fyuA-0-4S Treatment:standard growth conditions RAW_FILE_NAME=fyuA-0-4S SUBJECT_SAMPLE_FACTORS - fyuA-0-5S Treatment:standard growth conditions RAW_FILE_NAME=fyuA-0-5S SUBJECT_SAMPLE_FACTORS - fyuA-0-6S Treatment:standard growth conditions RAW_FILE_NAME=fyuA-0-6S SUBJECT_SAMPLE_FACTORS - fur-0-1S Treatment:No iron supplementation RAW_FILE_NAME=fur-0-1S SUBJECT_SAMPLE_FACTORS - fur-0-2S Treatment:No iron supplementation RAW_FILE_NAME=fur-0-2S SUBJECT_SAMPLE_FACTORS - fur-0-3S Treatment:No iron supplementation RAW_FILE_NAME=fur-0-3S SUBJECT_SAMPLE_FACTORS - fur-0-4S Treatment:No iron supplementation RAW_FILE_NAME=fur-0-4S SUBJECT_SAMPLE_FACTORS - fur-0-5S Treatment:No iron supplementation RAW_FILE_NAME=fur-0-5S SUBJECT_SAMPLE_FACTORS - fur-0-6S Treatment:No iron supplementation RAW_FILE_NAME=fur-0-6S #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 siderophores. 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 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 fur-0-1S fur-0-2S fur-0-3S fur-0-4S fur-0-5S fur-0-6S fyuA-0-1S fyuA-0-2S fyuA-0-3S fyuA-0-4S fyuA-0-5S fyuA-0-6S ybtP-0-1S ybtP-0-2S ybtP-0-3S ybtP-0-4S ybtP-0-5S ybtP-0-6S ybtQ-0-1S ybtQ-0-2S ybtQ-0-3S ybtQ-0-4S ybtQ-0-5S ybtQ-0-6S 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 enterobactin 418262 723051 881082 1047707 1106791 1064516 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 yersiniabactin 42329199 37395309 33396088 53337424 48843702 20929925 20696349 22632626 14746983 18704218 24716392 27709710 24676201 17235929 31427469 24862846 38203661 35885605 33972788 43217684 43029294 32819250 57602883 54175107 HPTT 98066429 91710913 83230824 113723527 111329679 70079936 66143013 76561851 51428167 60401047 65778788 72989442 105409290 109980050 125399554 117934855 130395991 133325450 108305608 107090119 116291344 103911416 133130879 130093650 salmochelin 187052 182890 143313 239876 167729 156563 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 yersiniabactin+Fe 0 0 0 0 0 0 101887261 113237323 79509533 104460098 127279618 146047210 91172175 68725164 116129287 93412622 135248148 129491365 102163848 131027612 126618756 101561241 158928389 160395185 HPTT-Fe 0 0 0 0 0 0 2150097 2694069 2474538 3051478 5864364 6795159 5434145 5874220 10424444 6758298 1128617 10817978 5264483 5331961 8801005 7479140 11941102 11597832 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name quantified m/z retention time enterobactin 670.1652 16.42 yersiniabactin 482.1342 17.92 HPTT 307.0206 18.22 salmochelin 404.1182 19.24 yersiniabactin+Fe 535.0356 15.4 HPTT-Fe 665.9588 18.12 METABOLITES_END #END