#METABOLOMICS WORKBENCH Codreags00_20250509_100344 DATATRACK_ID:5900 STUDY_ID:ST003919 ANALYSIS_ID:AN006435 PROJECT_ID:PR002452
VERSION             	1
CREATED_ON             	May 16, 2025, 8:40 am
#PROJECT
PR:PROJECT_TITLE                 	An anaerobic pathogen operates oxidative metabolism to colonize the inflamed
PR:PROJECT_TITLE                 	intestine
PR:PROJECT_TYPE                  	Untargeted Metabolomics analysis
PR:PROJECT_SUMMARY               	In contrast to the facultative pathogens, ETBF is canonically classified as an
PR:PROJECT_SUMMARY               	obligate anaerobe that is susceptible to oxygen toxicity and reliant on
PR:PROJECT_SUMMARY               	anaerobic fermentation for energy production. How ETBF successfully colonizes
PR:PROJECT_SUMMARY               	the inflamed gut, an environment enriched in oxygen and inflammatory
PR:PROJECT_SUMMARY               	mediators46, has remained unclear. Here, we demonstrate that ETBF leverages its
PR:PROJECT_SUMMARY               	virulence factor BFT to reprogram epithelial cell metabolism, thereby reshaping
PR:PROJECT_SUMMARY               	the gut nutritional landscape. This reprogramming leads to increased levels of
PR:PROJECT_SUMMARY               	lactate and oxygen, which fuel ETBF's unique oxidative metabolism, challenging
PR:PROJECT_SUMMARY               	the long-standing view of B. fragilis as strictly anaerobic. Our findings
PR:PROJECT_SUMMARY               	uncover a novel mechanism by which an obligate anaerobe reshapes its environment
PR:PROJECT_SUMMARY               	to establish an oxidative metabolic niche and promote its own expansion in the
PR:PROJECT_SUMMARY               	inflamed gut.
PR:INSTITUTE                     	Vanderbilt University
PR:DEPARTMENT                    	Chemistry
PR:LABORATORY                    	Center for Innovative Technology
PR:LAST_NAME                     	CODREANU
PR:FIRST_NAME                    	SIMONA Gabriela
PR:ADDRESS                       	1234 STEVENSON CENTER LANE
PR:EMAIL                         	SIMONA.CODREANU@VANDERBILT.EDU
PR:PHONE                         	6158758422
#STUDY
ST:STUDY_TITLE                   	An anaerobic pathogen operates oxidative metabolism to colonize the inflamed
ST:STUDY_TITLE                   	intestine
ST:STUDY_TYPE                    	untargeted metabolomics analysis
ST:STUDY_SUMMARY                 	To colonize the densely populated large intestine and cause disease, enteric
ST:STUDY_SUMMARY                 	pathogens must deploy their virulence factors to establish distinct nutrient
ST:STUDY_SUMMARY                 	niches. While facultative anaerobes exploit inflammation-induced oxygenation to
ST:STUDY_SUMMARY                 	gain a competitive advantage, how obligate anaerobic pathogens construct
ST:STUDY_SUMMARY                 	nutrient niches for colonization remains poorly understood. Here we show that
ST:STUDY_SUMMARY                 	enterotoxigenic Bacteroides fragilis (ETBF) an obligate anaerobe implicated in
ST:STUDY_SUMMARY                 	colitis and colorectal cancer, uses its virulence factor, Bacteroides fragilis
ST:STUDY_SUMMARY                 	toxin (BFT), to reprogram intestinal epithelial cell metabolism. BFT activates
ST:STUDY_SUMMARY                 	pro-tumorigenic signaling and hijacks the host bile acid recycling pathway,
ST:STUDY_SUMMARY                 	inducing a metabolic shift in the epithelium from oxidative phosphorylation to
ST:STUDY_SUMMARY                 	glycolysis. This shift increases intestinal lactate and oxygen, nutrients that
ST:STUDY_SUMMARY                 	fuel ETBF’s own oxidative metabolism. These findings reveal an unexpected
ST:STUDY_SUMMARY                 	strategy by which an obligate anaerobe generates and exploits an oxidative niche
ST:STUDY_SUMMARY                 	in the inflamed gut, redefining how microbial virulence factors shape host
ST:STUDY_SUMMARY                 	metabolism to promote pathogen persistence.
ST:INSTITUTE                     	Vanderbilt University
ST:DEPARTMENT                    	Chemistry
ST:LABORATORY                    	Center for Innovative Technology
ST:LAST_NAME                     	CODREANU
ST:FIRST_NAME                    	SIMONA Gabriela
ST:ADDRESS                       	1234 STEVENSON CENTER LANE
ST:EMAIL                         	SIMONA.CODREANU@VANDERBILT.EDU
ST:PHONE                         	16158758422
ST:NUM_GROUPS                    	2
ST:TOTAL_SUBJECTS                	8
#SUBJECT
SU:SUBJECT_TYPE                  	Mammal
SU:SUBJECT_SPECIES               	Mus musculus
SU:TAXONOMY_ID                   	ApcMin mice
SU:GENDER                        	Not applicable
#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           	-	8_01	Sample source:Fecal Content | genotype:Control	RAW_FILE_NAME(MS Name)=SC_20211201_RPLCn_FMS_Zhu_8_01
SUBJECT_SAMPLE_FACTORS           	-	8_02	Sample source:Fecal Content | genotype:Control	RAW_FILE_NAME(MS Name)=SC_20211201_RPLCn_FMS_Zhu_8_02
SUBJECT_SAMPLE_FACTORS           	-	8_03	Sample source:Fecal Content | genotype:Control	RAW_FILE_NAME(MS Name)=SC_20211201_RPLCn_FMS_Zhu_8_03
SUBJECT_SAMPLE_FACTORS           	-	8_04	Sample source:Fecal Content | genotype:Control	RAW_FILE_NAME(MS Name)=SC_20211201_RPLCn_FMS_Zhu_8_04
SUBJECT_SAMPLE_FACTORS           	-	9_01	Sample source:Fecal Content | genotype:Tumor	RAW_FILE_NAME(MS Name)=SC_20211201_RPLCn_FMS_Zhu_9_01
SUBJECT_SAMPLE_FACTORS           	-	9_02	Sample source:Fecal Content | genotype:Tumor	RAW_FILE_NAME(MS Name)=SC_20211201_RPLCn_FMS_Zhu_9_02
SUBJECT_SAMPLE_FACTORS           	-	9_03	Sample source:Fecal Content | genotype:Tumor	RAW_FILE_NAME(MS Name)=SC_20211201_RPLCn_FMS_Zhu_9_03
SUBJECT_SAMPLE_FACTORS           	-	9_04	Sample source:Fecal Content | genotype:Tumor	RAW_FILE_NAME(MS Name)=SC_20211201_RPLCn_FMS_Zhu_9_04
#COLLECTION
CO:COLLECTION_SUMMARY            	Notably, ETBF-infected ApcMin mice develop colonic tumors within weeks, well
CO:COLLECTION_SUMMARY            	before small intestinal adenomas are clinically evident or detectable.
CO:COLLECTION_SUMMARY            	Untargeted metabolomic profiling was performed on intestinal contents from
CO:COLLECTION_SUMMARY            	ETBF-infected mice twelve weeks post-infection.
CO:COLLECTION_PROTOCOL_FILENAME  	Fecal_Samples.pdf
CO:SAMPLE_TYPE                   	Feces
CO:STORAGE_CONDITIONS            	-80℃
#TREATMENT
TR:TREATMENT_SUMMARY             	Notably, ETBF-infected ApcMin mice develop colonic tumors within weeks, well
TR:TREATMENT_SUMMARY             	before small intestinal adenomas are clinically evident or detectable. ApcMin
TR:TREATMENT_SUMMARY             	mice were colonized with the ETBF strains and received a single dose of
TR:TREATMENT_SUMMARY             	tributyrin or a mock control. Samples were collected 12 weeks post-inoculation.
TR:TREATMENT_SUMMARY             	Twelve weeks post-infection, we assessed colonic tumor incidence. These findings
TR:TREATMENT_SUMMARY             	support a model in which ETBF-induced tumorigenesis is potentiated by its
TR:TREATMENT_SUMMARY             	capacity to exploit oxygen and engage in oxidative metabolism, suggesting a link
TR:TREATMENT_SUMMARY             	between microbial respiration and cancer promotion in the gut.
TR:TREATMENT_PROTOCOL_FILENAME   	Fecal_Samples.pdf
TR:TREATMENT_COMPOUND            	tributyrin
#SAMPLEPREP
SP:SAMPLEPREP_SUMMARY            	Samples were stored at -80°C until analyzed via Liquid Chromatography-High
SP:SAMPLEPREP_SUMMARY            	Resolution Mass Spectrometry (LC-HRMS and LC-HRMS/MS)-based metabolomics in the
SP:SAMPLEPREP_SUMMARY            	Vanderbilt Center for Innovative Technology (CIT) using previously described
SP:SAMPLEPREP_SUMMARY            	methods (PMID: 34068340 PMID: 27955696 PMID: 29774083). Briefly, frozen mouse
SP:SAMPLEPREP_SUMMARY            	intestinal content samples (n=8, 4 biological replicates for each sample groups)
SP:SAMPLEPREP_SUMMARY            	were lysed in 1000 µl ice-cold lysis buffer (1:1:2,v:v:v, acetonitrile:
SP:SAMPLEPREP_SUMMARY            	methanol: ammonium bicarbonate 0.1M - pH 8.0) and sonicated individually using a
SP:SAMPLEPREP_SUMMARY            	probe tip sonicator at 50% power (10 pulses). Homogenized samples were
SP:SAMPLEPREP_SUMMARY            	normalized by weight to the smallest amount of tissue sample to an equal amount
SP:SAMPLEPREP_SUMMARY            	per sample. Proteins were precipitated from individual samples by addition of
SP:SAMPLEPREP_SUMMARY            	800 µL of ice-cold methanol followed by overnight incubation at -80°C.
SP:SAMPLEPREP_SUMMARY            	Precipitated proteins were pelleted by centrifugation (15k rpm, 15 min) and
SP:SAMPLEPREP_SUMMARY            	metabolite extracts were dried down in vacuo. Individual extracts were
SP:SAMPLEPREP_SUMMARY            	reconstituted in 100 µl of acetonitrile/water (3:97, v/v) with 0.1% formic acid
SP:SAMPLEPREP_SUMMARY            	containing heavy-labeled carnitine-D9, tryptophan-D3, valine-D8, and
SP:SAMPLEPREP_SUMMARY            	inosine-4N15, and centrifuged for 5 min at 10,000 rpm to remove insoluble
SP:SAMPLEPREP_SUMMARY            	material. A pooled quality control sample (QC) was prepared by pooling equal
SP:SAMPLEPREP_SUMMARY            	volumes of individual samples. The pooled QC sample was used for column
SP:SAMPLEPREP_SUMMARY            	conditioning (8 injections prior to sample analysis), retention time alignment
SP:SAMPLEPREP_SUMMARY            	and to assess mass spectrometry instrument reproducibility throughout the sample
SP:SAMPLEPREP_SUMMARY            	set and to determine sample acceptance.
SP:SAMPLEPREP_PROTOCOL_FILENAME  	Metabolomics_Methods.pdf
SP:PROCESSING_STORAGE_CONDITIONS 	-80℃
SP:EXTRACT_STORAGE               	-80℃
#CHROMATOGRAPHY
CH:CHROMATOGRAPHY_SUMMARY        	RPLC positive - hydrophobic compounds
CH:CHROMATOGRAPHY_TYPE           	Reversed phase
CH:INSTRUMENT_NAME               	Thermo Vanquish
CH:COLUMN_NAME                   	Thermo Hypersil GOLD aQ (100 x 2.1mm,1.9um)
CH:SOLVENT_A                     	100% water; 0.1% formic acid;
CH:SOLVENT_B                     	80% Acetonitrile; 20% water; 0.1% formic acid;
CH:FLOW_GRADIENT                 	Linear gradient from 0-1 min 1% B, 1-10min 35%B, 10-15min 70%B, 15-19min 99%B,
CH:FLOW_GRADIENT                 	19-25min 99%B, 25-25.5min 1%B, 25.5-30min 1%B
CH:FLOW_RATE                     	0.25 mL/min
CH:COLUMN_TEMPERATURE            	40
CH:METHODS_FILENAME              	Metabolomics_Methods.pdf
#ANALYSIS
AN:ANALYSIS_TYPE                 	MS
AN:ANALYSIS_PROTOCOL_FILE        	Metabolomics_Methods.pdf
#MS
MS:INSTRUMENT_NAME               	Thermo Q Exactive Orbitrap
MS:INSTRUMENT_TYPE               	Orbitrap
MS:MS_TYPE                       	Other
MS:ION_MODE                      	POSITIVE
MS:MS_COMMENTS                   	reverse phase positive mode Progenesis QI
MS:MS_RESULTS_FILE               	ST003919_AN006435_Results.txt	UNITS:Peak intensity	Has m/z:Yes	Has RT:Yes	RT units:Minutes
#END