#METABOLOMICS WORKBENCH Lu_Group_20220325_181827 DATATRACK_ID:3141 STUDY_ID:ST002131 ANALYSIS_ID:AN003485 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                      	POSITIVE
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
L-Proline	986.789907	3216.09878	4028.54439	3677.30929	3269.49938	2563.7473	18329.6334	4132.50612	2821.82694	1952.54697	2395.56784	6191.11024	4182.60924	7211.2887	3982.68581	3841.28957	2666.90632	3721.40127	19069.6256	29679.8785	24668.8744	18464.284	16546.817	28479.8744
L-Glutamate	7526.1027	7498.23206	6636.29775	9334.26861	6083.34986	6224.92632	4769923.08	3594.15505	3193.45563	2879.58426	2596.76919	3640.7453	11277.6297	11891.3347	16301.8768	13295.2443	14914.1068	16171.2394	18370.5821	30178.0965	29215.1653	18421.8643	25950.4105	26076.1513
L-Aspartic acid	164.124147	191.422551	225.402983	253.895024	156.191251	151.086897	24838.4744	134.009375	108.521457	107.539062	123.677803	172.932173	263.659024	303.578058	286.521193	229.027772	263.401424	248.907545	179.570251	260.193418	278.042525	197.159045	213.099744	272.499526
Deoxycytidine	295.225627	136.631887	243.132093	275.765958	204.796461	192.758744	197.332525	136.903638	107.691271	107.82168	92.3542544	125.119394	15.2698091	34.249451	19.9925231	14.7894682	13.6995848	24.3218509	187.273049	112.605741	139.799984	175.882943	261.930844	154.171312
L-Citrulline	88.4233014	88.7245745	90.3825403	65.9646174	87.8613747	88.7408443	161.991533	140.453983	151.472324	106.385096	105.1525	169.410165	142.761777	175.846944	150.91422	167.76002	161.522968	162.54848	167.189166	214.140154	246.386832	141.054067	223.298112	302.466006
Cytidine	527.784321	555.675564	939.686213	653.239145	873.964991	781.124551	428.974606	341.059406	458.847517	453.777827	368.268993	511.976216	114.984441	237.130577	199.080297	165.04016	167.539961	174.194419	186.901017	198.723451	200.268576	144.158759	178.900449	185.358683
Histamine	3864.51011	3496.06222	4901.3858	2810.69608	3832.43616	3916.13417	2375.7933	1934.94726	2674.76119	1696.36893	1302.39202	2975.04108	1454.12579	2119.99269	1388.54084	1558.64915	1404.6812	1358.89233	3188.71908	5081.43049	4150.83134	3233.18006	5164.04832	6922.63658
L-Arginine	229.302681	2456.63053	3962.11253	569.464316	6228.20053	1277.57321	7083.65897	316.736842	1733.07657	1491.59143	1595.6016	1890.54122	4201.48066	6610.60363	1275.36642	2586.35737	2914.21529	2479.92051	1910.86375	3150.56285	3211.11407	1848.80529	2368.46815	4064.23768
L-Lysine	4811.26587	3521.93985	3430.66755	3018.52635	3103.93657	2325.05325	10307.7776	8620.99596	6801.58465	6923.94055	6620.7342	8186.41831	13067.8305	12714.4747	6302.73657	7386.64873	6842.69484	6570.00543	8320.666	5811.96575	8257.50706	7275.23583	9682.95682	9013.644
Thymine	30674.244	18636.7838	18795.524	28281.5032	17433.574	27396.6158	17890.6273	10377.4891	18758.1211	22766.369	20245.6705	25776.9367	18778.1543	34215.875	32392.2441	33870.7831	23757.6055	31568.3548	14264.3484	20170.1725	12068.3172	12428.0649	14486.5873	27740.7073
5-aminosalicylic acid	11639.2205	4660.82707	6200.60246	11982.1976	6370.08613	6077.03637	3977.47619	5113.78554	3983.67186	8558.65351	7715.00948	4320.15745	10451.0472	9856.56993	11658.9732	12782.4671	13577.9836	16154.2958	11314.1014	8235.74654	9214.29719	9799.30503	10446.448	11406.4405
Adenosine 5'-monophosphate	12156.6092	73911.0684	107335.977	106674.301	140930.379	110248.427	59379.7668	50389.9073	55512.1303	52942.5323	68508.4783	142113.541	207981.665	279580.38	238193.637	194293.576	238358.882	269098.686	221427.861	240573.774	232050.337	244656.232	210098.54	242684.009
Cytosine	3911.1325	3496.06222	5234.22024	2678.6437	3832.43616	3916.13417	3552.27187	2484.97416	2674.76119	1682.74908	1625.01142	2975.04108	1454.12579	2119.99269	1981.42275	1558.64915	1451.80714	1629.68995	2464.75467	3695.64279	3281.03862	3078.69673	3666.2703	4222.93396
Tryptophan	357505.135	233797.713	210485.589	224664.664	259168.262	391194.876	354602.368	246181.519	349933.453	467050.69	297886.853	343728.965	302846.062	325622.971	389605.605	299335.021	318201.328	331713.104	290811.03	301569.31	304623.81	382698.154	402823.595	346217.616
Adenine	218484.973	119618.775	147390.954	165672.936	160387.905	136168.854	196109.696	116331.834	121834.714	138370.97	111136.524	134878.086	20995.094	52740.516	71180.0215	41298.6867	32444.0507	39315.787	32186.738	28649.4561	31485.4668	31201.4297	40490.5648	41069.7763
L-Ornithine	2769.60171	1612.86279	1663.15534	2051.09114	1498.66571	1451.92083	3112.02878	2024.51771	2014.39996	3617.77284	3746.1313	3645.12869	2322.95023	3317.62779	4163.06231	3359.90998	2456.57507	3257.63051	3055.38317	2232.89193	2749.25971	3249.64668	3478.36197	2962.56842
MS_METABOLITE_DATA_END
#METABOLITES
METABOLITES_START
metabolite_name	Molecular weight	RT (min)	m/z
L-Proline	115.13	1	116
L-Glutamate	147.13	0.9	148
L-Aspartic acid	133.1	1.05	134.11
Deoxycytidine	227.22	1.5	228
L-Citrulline	175.19	0.94	176.2
Cytidine	243.22	1.3	244
Histamine	111.07	1	112
L-Arginine	174.2	0.96	175.21
L-Lysine	146.19	0.8	147.2
Thymine	126.11	2.92	127.12
5-aminosalicylic acid	153.14	1.54	154.15
Adenosine 5'-monophosphate	347.22	1.51	348.07
Cytosine	111.1	1.5	112
Adenine	135.13	1.5	136
L-Ornithine	132.19	0.78	133.1
Tryptophan	204.23	5.71	205.1
METABOLITES_END
#END