#METABOLOMICS WORKBENCH ARutledge1_20190814_111842 DATATRACK_ID:1803 STUDY_ID:ST001244 ANALYSIS_ID:AN002067 PROJECT_ID:PR000831
VERSION             	1
CREATED_ON             	August 20, 2019, 2:08 pm
#PROJECT
PR:PROJECT_TITLE                 	Uropathogenic versus Urocolonizing Escherichia coli
PR:PROJECT_SUMMARY               	Urinary tract infections (UTIs) represent a major burden across the population,
PR:PROJECT_SUMMARY               	although key facets of their pathogenesis challenge physicians and investigators
PR:PROJECT_SUMMARY               	alike. Escherichia coli epitomizes these obstacles: this Gram-negative bacterial
PR:PROJECT_SUMMARY               	species is the most prevalent agent of UTIs worldwide and can also colonize the
PR:PROJECT_SUMMARY               	urogenital tract in a phenomenon known as asymptomatic bacteriuria (ASB).
PR:PROJECT_SUMMARY               	Unfortunately, at the level of the organism, the relationship between
PR:PROJECT_SUMMARY               	symptomatic UTI and ASB is poorly defined, confounding our understanding of
PR:PROJECT_SUMMARY               	microbial pathogenesis and strategies for clinical management. Unlike
PR:PROJECT_SUMMARY               	diarrheagenic pathotypes of E. coli, the definition of uropathogenic E. coli
PR:PROJECT_SUMMARY               	(UPEC) remains phenomenologic, without conserved phenotypes and (known) genetic
PR:PROJECT_SUMMARY               	determinants that rigorously distinguish UTI- and ASB-associated strains. This
PR:PROJECT_SUMMARY               	manuscript provides a cross-disciplinary review of the current issues – from
PR:PROJECT_SUMMARY               	interrelated mechanistic and diagnostic perspectives – and describes new
PR:PROJECT_SUMMARY               	opportunities by which clinical resources can be leveraged to overcome molecular
PR:PROJECT_SUMMARY               	challenges. Specifically, we present our work harnessing a large collection of
PR:PROJECT_SUMMARY               	patient-derived isolates to identify features that do (and do not) distinguish
PR:PROJECT_SUMMARY               	UTI- from ASB-associated E. coli strains. Analyses of biofilm formation,
PR:PROJECT_SUMMARY               	previously reported to be higher in ASB strains, revealed extensive phenotypic
PR:PROJECT_SUMMARY               	heterogeneity that did not correlate with symptomatology. However, metabolomic
PR:PROJECT_SUMMARY               	experiments revealed distinct signatures between ASB and cystitis isolates,
PR:PROJECT_SUMMARY               	including species in the purine pathway (previously shown to be critical for
PR:PROJECT_SUMMARY               	intracellular survival during acute infection). Together, these studies
PR:PROJECT_SUMMARY               	demonstrate how large-scale, wild-type approaches can help dissect the
PR:PROJECT_SUMMARY               	physiology of colonization-versus-infection, suggesting that the molecular
PR:PROJECT_SUMMARY               	definition of UPEC may rest at the level of global bacterial metabolism.
PR:INSTITUTE                     	Vanderbilt University
PR:LAST_NAME                     	Rutledge
PR:FIRST_NAME                    	Alexandra
PR:ADDRESS                       	7330 Stevenson Center Lane, NASHVILLE, TENNESSEE, 37235, USA
PR:EMAIL                         	a.rutledge@vanderbilt.edu
PR:PHONE                         	6155046923
#STUDY
ST:STUDY_TITLE                   	Uropathogenic versus Urocolonizing Escherichia coli
ST:STUDY_SUMMARY                 	Urinary tract infections (UTIs) represent a major burden across the population,
ST:STUDY_SUMMARY                 	although key facets of their pathogenesis challenge physicians and investigators
ST:STUDY_SUMMARY                 	alike. Escherichia coli epitomizes these obstacles: this Gram-negative bacterial
ST:STUDY_SUMMARY                 	species is the most prevalent agent of UTIs worldwide and can also colonize the
ST:STUDY_SUMMARY                 	urogenital tract in a phenomenon known as asymptomatic bacteriuria (ASB).
ST:STUDY_SUMMARY                 	Unfortunately, at the level of the organism, the relationship between
ST:STUDY_SUMMARY                 	symptomatic UTI and ASB is poorly defined, confounding our understanding of
ST:STUDY_SUMMARY                 	microbial pathogenesis and strategies for clinical management. Unlike
ST:STUDY_SUMMARY                 	diarrheagenic pathotypes of E. coli, the definition of uropathogenic E. coli
ST:STUDY_SUMMARY                 	(UPEC) remains phenomenologic, without conserved phenotypes and (known) genetic
ST:STUDY_SUMMARY                 	determinants that rigorously distinguish UTI- and ASB-associated strains. This
ST:STUDY_SUMMARY                 	manuscript provides a cross-disciplinary review of the current issues – from
ST:STUDY_SUMMARY                 	interrelated mechanistic and diagnostic perspectives – and describes new
ST:STUDY_SUMMARY                 	opportunities by which clinical resources can be leveraged to overcome molecular
ST:STUDY_SUMMARY                 	challenges. Specifically, we present our work harnessing a large collection of
ST:STUDY_SUMMARY                 	patient-derived isolates to identify features that do (and do not) distinguish
ST:STUDY_SUMMARY                 	UTI- from ASB-associated E. coli strains. Analyses of biofilm formation,
ST:STUDY_SUMMARY                 	previously reported to be higher in ASB strains, revealed extensive phenotypic
ST:STUDY_SUMMARY                 	heterogeneity that did not correlate with symptomatology. However, metabolomic
ST:STUDY_SUMMARY                 	experiments revealed distinct signatures between ASB and cystitis isolates,
ST:STUDY_SUMMARY                 	including species in the purine pathway (previously shown to be critical for
ST:STUDY_SUMMARY                 	intracellular survival during acute infection). Together, these studies
ST:STUDY_SUMMARY                 	demonstrate how large-scale, wild-type approaches can help dissect the
ST:STUDY_SUMMARY                 	physiology of colonization-versus-infection, suggesting that the molecular
ST:STUDY_SUMMARY                 	definition of UPEC may rest at the level of global bacterial metabolism.
ST:INSTITUTE                     	Vanderbilt University
ST:LAST_NAME                     	Rutledge
ST:FIRST_NAME                    	Alexandra
ST:ADDRESS                       	7330 Stevenson Center Lane, NASHVILLE, TENNESSEE, 37235, USA
ST:EMAIL                         	a.rutledge@vanderbilt.edu
ST:PHONE                         	6155046923
#SUBJECT
SU:SUBJECT_TYPE                  	Bacteria
SU:SUBJECT_SPECIES               	Escherichia coli
SU:TAXONOMY_ID                   	562
SU:GENDER                        	Not applicable
#SUBJECT_SAMPLE_FACTORS:         	SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data
SUBJECT_SAMPLE_FACTORS           	-	SC_20180803_RPLCp_DDA2_Sup_QC_01	Group:Qcpool_1	
SUBJECT_SAMPLE_FACTORS           	-	SC_20180803_RPLCp_DDA2_Sup_QC_06	Group:Qcpool_1	
SUBJECT_SAMPLE_FACTORS           	-	SC_20180803_RPLCp_DDA4_Sup_QC_02	Group:Qcpool_1	
SUBJECT_SAMPLE_FACTORS           	-	SC_20180803_RPLCp_DDA4_Sup_QC_08	Group:Qcpool_1	
SUBJECT_SAMPLE_FACTORS           	-	SC_20180803_RPLCp_DDA6_Sup_QC_03	Group:Qcpool_1	
SUBJECT_SAMPLE_FACTORS           	-	SC_20180803_RPLCp_DDA6_Sup_QC_10	Group:Qcpool_1	
SUBJECT_SAMPLE_FACTORS           	-	SC_20180803_RPLCp_FMS_Sup_QC_04	Group:Qcpool_1	
SUBJECT_SAMPLE_FACTORS           	-	SC_20180803_RPLCp_FMS_Sup_QC_05	Group:Qcpool_1	
SUBJECT_SAMPLE_FACTORS           	-	SC_20180803_RPLCp_FMS_Sup_QC_07	Group:Qcpool_1	
SUBJECT_SAMPLE_FACTORS           	-	SC_20180803_RPLCp_FMS_Sup_QC_09	Group:Qcpool_1	
SUBJECT_SAMPLE_FACTORS           	-	SC_20180803_RPLCp_FMS_Sup_QC_11	Group:Qcpool_1	
SUBJECT_SAMPLE_FACTORS           	-	SC_20180803_RPLCp_FMS_Sup_S1_A1	Group:ASB_1	
SUBJECT_SAMPLE_FACTORS           	-	SC_20180803_RPLCp_FMS_Sup_S2_B1	Group:ASB_1	
SUBJECT_SAMPLE_FACTORS           	-	SC_20180803_RPLCp_FMS_Sup_S3_C1	Group:ASB_1	
SUBJECT_SAMPLE_FACTORS           	-	SC_20180803_RPLCp_FMS_Sup_S4_D1	Group:Cystitis_1	
SUBJECT_SAMPLE_FACTORS           	-	SC_20180803_RPLCp_FMS_Sup_S5_E1	Group:Cystitis_1	
SUBJECT_SAMPLE_FACTORS           	-	SC_20180803_RPLCp_FMS_Sup_S6_F1	Group:Cystitis_1	
SUBJECT_SAMPLE_FACTORS           	-	SC_20180803_RPLCp_FMS_Sup_S7_G1	Group:Cystitis_1	
SUBJECT_SAMPLE_FACTORS           	-	SC_20180803_RPLCp_FMS_Sup_S8_H1	Group:Cystitis_1	
SUBJECT_SAMPLE_FACTORS           	-	SC_20180803_RPLCp_FMS_Sup_S9_I1	Group:Cystitis_1	
SUBJECT_SAMPLE_FACTORS           	-	SC_20180803_RPLCp_FMS_Sup_S10_J1	Group:Cystitis_1	
SUBJECT_SAMPLE_FACTORS           	-	SC_20180803_RPLCp_FMS_Sup_S11_K1	Group:Cystitis_1	
SUBJECT_SAMPLE_FACTORS           	-	SC_20180803_RPLCp_FMS_Sup_S12_L1	Group:Cystitis_1	
#COLLECTION
CO:COLLECTION_SUMMARY            	Urine-associated E. coli isolates were collected in accordance with an approved
CO:COLLECTION_SUMMARY            	IRB
CO:SAMPLE_TYPE                   	Bacterial cells
#TREATMENT
TR:TREATMENT_SUMMARY             	For each isolate, a single colony from an agar dish was inoculated in 5 mL LB
TR:TREATMENT_SUMMARY             	and shaken overnight at 37°C under ambient atmospheric conditions. Cultures
TR:TREATMENT_SUMMARY             	were then diluted 1:1000 in the combined human urine and grown for 6 hours to
TR:TREATMENT_SUMMARY             	mid-log phase (37°C, shaking), under 4% oxygen to emulate the bladder
TR:TREATMENT_SUMMARY             	environment. After 6 hours, OD600 of each isolate was measured – and cultures
TR:TREATMENT_SUMMARY             	were normalized by volume to yield equal number of organisms from each strain
TR:TREATMENT_SUMMARY             	– prior to pooling into groups of 8 isolates each. CFUs were enumerated for
TR:TREATMENT_SUMMARY             	each pool to confirm bacterial denisty (~109 total E. coli per pool). Each pool
TR:TREATMENT_SUMMARY             	was then centrifuged to separate the cellular (pellet) and supernatant fraction.
TR:TREATMENT_SUMMARY             	Pellets and supernatants were flash frozen and stored at -80°C until for
TR:TREATMENT_SUMMARY             	metabolomic analysis.
#SAMPLEPREP
SP:SAMPLEPREP_SUMMARY            	Global untargeted metabolomic analyses were performed on the
SP:SAMPLEPREP_SUMMARY            	supernatant-fraction of ASB and cystitis pools. Aliquots of each pool (200µL)
SP:SAMPLEPREP_SUMMARY            	were added to individual Eppendorf tubes containing 200µL ice cold lysis buffer
SP:SAMPLEPREP_SUMMARY            	(1:1:2, ACN:MeOH:Ammonium Bicarbonate (0.1M, pH 8.0)) (LC-MS grade). Labeled
SP:SAMPLEPREP_SUMMARY            	creatinine-D3 and lysine-D4 were added to each sample to assess the metabolite
SP:SAMPLEPREP_SUMMARY            	extraction (sample preparation) step. Samples were first subjected to protein
SP:SAMPLEPREP_SUMMARY            	precipitation by addition of 800µL of ice cold methanol (4x by volume), then
SP:SAMPLEPREP_SUMMARY            	incubated at -80C overnight. Following incubation, samples were centrifuged
SP:SAMPLEPREP_SUMMARY            	(10,000 rpm, 10 min) to pellet precipitated proteins; the metabolite-containing
SP:SAMPLEPREP_SUMMARY            	supernatant was transferred to a clean Eppendorf tube, dried in vacuo and stored
SP:SAMPLEPREP_SUMMARY            	at -80C until further LC-MS analysis. The pellet-fraction of each sample pool
SP:SAMPLEPREP_SUMMARY            	prepared as described above was lysed using 400µL ice cold lysis buffer (1:1:2,
SP:SAMPLEPREP_SUMMARY            	ACN:MeOH:Ammonium Bicarbonate (0.1M, pH 8.0) (LC-MS grade), followed by
SP:SAMPLEPREP_SUMMARY            	sonication in an ice bath for 10 min. Sample volume for each pool was adjusted
SP:SAMPLEPREP_SUMMARY            	such that all samples have the same cell number in each vial. Samples were first
SP:SAMPLEPREP_SUMMARY            	subjected to protein precipitation by addition of 1000µL of ice cold methanol
SP:SAMPLEPREP_SUMMARY            	(4x by volume), then incubated at -80C overnight. Following incubation,
SP:SAMPLEPREP_SUMMARY            	samplwere were centrifuged (10,000 rpm, 10 min) to pellet precipitated proteins;
SP:SAMPLEPREP_SUMMARY            	the metabolite-containing extract was transferred to a clean Eppendorf tube,
SP:SAMPLEPREP_SUMMARY            	dried in vacuo and stored at -80C until further LC-MS analysis.
#CHROMATOGRAPHY
CH:CHROMATOGRAPHY_TYPE           	Reversed phase
CH:INSTRUMENT_NAME               	Thermo Vanquish
CH:COLUMN_NAME                   	Thermo Hypersil Gold
#ANALYSIS
AN:ANALYSIS_TYPE                 	MS
#MS
MS:INSTRUMENT_NAME               	Thermo Q Exactive HF hybrid Orbitrap
MS:INSTRUMENT_TYPE               	Orbitrap
MS:MS_TYPE                       	ESI
MS:ION_MODE                      	POSITIVE
MS:MS_COMMENTS                   	Progenesis QI
MS:MS_RESULTS_FILE               	ST001244_AN002067_Results.txt	UNITS:abundance	Has m/z:Yes	Has RT:Yes	RT units:Minutes
#END