#METABOLOMICS WORKBENCH Codreags00_20250317_090522 DATATRACK_ID:5728 STUDY_ID:ST003796 ANALYSIS_ID:AN006241 PROJECT_ID:PR002371 VERSION 1 CREATED_ON March 17, 2025, 9:32 am #PROJECT PR:PROJECT_TITLE Short-term alterations in dietary amino acids override host genetic PR:PROJECT_TITLE susceptibility and reveal mechanisms of Salmonella Typhimurium small intestine PR:PROJECT_TITLE colonization PR:PROJECT_TYPE Untargeted Metabolomics analysis PR:PROJECT_SUMMARY In addition to individual genetics, environmental factors, i.e., short-term PR:PROJECT_SUMMARY dietary changes, may influence host susceptibility to gastrointestinal PR:PROJECT_SUMMARY infection. Herein, we developed a model in which CBA/J mice, a genetically PR:PROJECT_SUMMARY resistant strain that tolerates intestinal colonization by the enteric pathogen PR:PROJECT_SUMMARY Salmonella Typhimurium (S. Tm), rapidly succumb to infectious gastroenteritis PR:PROJECT_SUMMARY after exposure to a L-amino acids (AA) rich diet. In mice, S. Tm-gastroenteritis PR:PROJECT_SUMMARY is restricted to the cecum (large intestine), limiting their use to understand PR:PROJECT_SUMMARY S. Tm small intestine (ileum) colonization, a feature of human Salmonellosis. PR:PROJECT_SUMMARY Surprisingly, CBA mice fed AA diet demonstrated enhanced S. Tm ileal PR:PROJECT_SUMMARY colonization and mortality. Using germ-free mice and ileal-fecal slurry PR:PROJECT_SUMMARY transplant, we found that diet-dependent S. Tm ileal colonization to be PR:PROJECT_SUMMARY microbiota-dependent. Mechanistically, S. Tm relied on Fructosyl-asparagine PR:PROJECT_SUMMARY utilization to expand in the ileum during infection. We demonstrate how AA-based PR:PROJECT_SUMMARY diet overrides host genetics by altering the gut microbiota’s ability to PR:PROJECT_SUMMARY prevent S. Tm ileal colonization. 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 Short-term alterations in dietary amino acids override host genetic ST:STUDY_TITLE susceptibility and reveal mechanisms of Salmonella Typhimurium small intestine ST:STUDY_TITLE colonization ST:STUDY_TYPE untargeted metabolomics analysis ST:STUDY_SUMMARY Herein, we find that alterations in dietary amino acid influence the ST:STUDY_SUMMARY susceptibility of genetically resistant CBA/J mice to infection by S. Tm. ST:STUDY_SUMMARY Interestingly, increased susceptibility is not due to the overall inability of ST:STUDY_SUMMARY the murine host to handle infection or differences in host transcriptome. ST:STUDY_SUMMARY Significant reductions in microbial diversity of the small intestine impact the ST:STUDY_SUMMARY ability of the small intestine microbiota to prevent colonization and expansion ST:STUDY_SUMMARY of the pathogen. Using this model, we identified small intestine-relevant ST:STUDY_SUMMARY metabolic pathways necessary for S. Tm expansion, such as using ST:STUDY_SUMMARY Fructosyl-Asparagine, which enables expansion in the terminal ileum. 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 6158758422 ST:NUM_GROUPS 3 ST:TOTAL_SUBJECTS 18 ST:NUM_MALES 9 ST:NUM_FEMALES 9 ST:STUDY_COMMENTS Significant reductions in microbial diversity of the small intestine impact the ST:STUDY_COMMENTS ability of the small intestine microbiota to prevent colonization and expansion ST:STUDY_COMMENTS of the pathogen. #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID CBA/J mice, a genetically resistant strain SU:AGE_OR_AGE_RANGE 6-7 weeks old for all CBA/J SU:GENDER Male and female SU:ANIMAL_ANIMAL_SUPPLIER Jackson Laboratories #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 36-1 I Met Diet2_CH_01 Sample source:Mouse Intestinal Content | Genotype:CBA/J mice | Treatment:Chow RAW_FILE_NAME(MS Sample Name)=SC_20221214_HILICp_FMS_Diet2_CH_01 SUBJECT_SAMPLE_FACTORS 36-2 I Met Diet2_CH_02 Sample source:Mouse Intestinal Content | Genotype:CBA/J mice | Treatment:Chow RAW_FILE_NAME(MS Sample Name)=SC_20221214_HILICp_FMS_Diet2_CH_02 SUBJECT_SAMPLE_FACTORS 36-3 I Met Diet2_CH_03 Sample source:Mouse Intestinal Content | Genotype:CBA/J mice | Treatment:Chow RAW_FILE_NAME(MS Sample Name)=SC_20221214_HILICp_FMS_Diet2_CH_03 SUBJECT_SAMPLE_FACTORS 36-4 I Met Diet2_CH_04 Sample source:Mouse Intestinal Content | Genotype:CBA/J mice | Treatment:Chow RAW_FILE_NAME(MS Sample Name)=SC_20221214_HILICp_FMS_Diet2_CH_04 SUBJECT_SAMPLE_FACTORS 36-5 I Met Diet2_CH_05 Sample source:Mouse Intestinal Content | Genotype:CBA/J mice | Treatment:Chow RAW_FILE_NAME(MS Sample Name)=SC_20221214_HILICp_FMS_Diet2_CH_05 SUBJECT_SAMPLE_FACTORS 36-6 I Met Diet2_CH_06 Sample source:Mouse Intestinal Content | Genotype:CBA/J mice | Treatment:Chow RAW_FILE_NAME(MS Sample Name)=SC_20221214_HILICp_FMS_Diet2_CH_06 SUBJECT_SAMPLE_FACTORS 36-7 I Met Diet2_AA_07 Sample source:Mouse Intestinal Content | Genotype:CBA/J mice | Treatment:AA Diet RAW_FILE_NAME(MS Sample Name)=SC_20221214_HILICp_FMS_Diet2_AA_07 SUBJECT_SAMPLE_FACTORS 36-8 I Met Diet2_AA_08 Sample source:Mouse Intestinal Content | Genotype:CBA/J mice | Treatment:AA Diet RAW_FILE_NAME(MS Sample Name)=SC_20221214_HILICp_FMS_Diet2_AA_08 SUBJECT_SAMPLE_FACTORS 36-9 I Met Diet2_AA_09 Sample source:Mouse Intestinal Content | Genotype:CBA/J mice | Treatment:AA Diet RAW_FILE_NAME(MS Sample Name)=SC_20221214_HILICp_FMS_Diet2_AA_09 SUBJECT_SAMPLE_FACTORS 36-10 I Met Diet2_AA_10 Sample source:Mouse Intestinal Content | Genotype:CBA/J mice | Treatment:AA Diet RAW_FILE_NAME(MS Sample Name)=SC_20221214_HILICp_FMS_Diet2_AA_10 SUBJECT_SAMPLE_FACTORS 36-11 I Met Diet2_AA_11 Sample source:Mouse Intestinal Content | Genotype:CBA/J mice | Treatment:AA Diet RAW_FILE_NAME(MS Sample Name)=SC_20221214_HILICp_FMS_Diet2_AA_11 SUBJECT_SAMPLE_FACTORS 36-12 I Met Diet2_AA_12 Sample source:Mouse Intestinal Content | Genotype:CBA/J mice | Treatment:AA Diet RAW_FILE_NAME(MS Sample Name)=SC_20221214_HILICp_FMS_Diet2_AA_12 SUBJECT_SAMPLE_FACTORS 36-13 I Met Diet2_CA_13 Sample source:Mouse Intestinal Content | Genotype:CBA/J mice | Treatment:Casein Diet RAW_FILE_NAME(MS Sample Name)=SC_20221214_HILICp_FMS_Diet2_CA_13 SUBJECT_SAMPLE_FACTORS 36-14 I Met Diet2_CA_14 Sample source:Mouse Intestinal Content | Genotype:CBA/J mice | Treatment:Casein Diet RAW_FILE_NAME(MS Sample Name)=SC_20221214_HILICp_FMS_Diet2_CA_14 SUBJECT_SAMPLE_FACTORS 36-15 I Met Diet2_CA_15 Sample source:Mouse Intestinal Content | Genotype:CBA/J mice | Treatment:Casein Diet RAW_FILE_NAME(MS Sample Name)=SC_20221214_HILICp_FMS_Diet2_CA_15 SUBJECT_SAMPLE_FACTORS 36-16 I Met Diet2_CA_16 Sample source:Mouse Intestinal Content | Genotype:CBA/J mice | Treatment:Casein Diet RAW_FILE_NAME(MS Sample Name)=SC_20221214_HILICp_FMS_Diet2_CA_16 SUBJECT_SAMPLE_FACTORS 36-17 I Met Diet2_CA_17 Sample source:Mouse Intestinal Content | Genotype:CBA/J mice | Treatment:Casein Diet RAW_FILE_NAME(MS Sample Name)=SC_20221214_HILICp_FMS_Diet2_CA_17 SUBJECT_SAMPLE_FACTORS 36-18 I Met Diet2_CA_18 Sample source:Mouse Intestinal Content | Genotype:CBA/J mice | Treatment:Casein Diet RAW_FILE_NAME(MS Sample Name)=SC_20221214_HILICp_FMS_Diet2_CA_18 #COLLECTION CO:COLLECTION_SUMMARY Both sexes were equally represented in each experimental group. Animals were CO:COLLECTION_SUMMARY randomly assigned into cages and treatment groups 3 days prior to CO:COLLECTION_SUMMARY experimentation. Unless stated otherwise, a minimum of 5 mice were used based on CO:COLLECTION_SUMMARY variability observed in previous experiments. All mice were monitored daily, and CO:COLLECTION_SUMMARY cage bedding changed every two weeks. At the end of the experiments, mice were CO:COLLECTION_SUMMARY humanely euthanized using carbon dioxide inhalation. Animals that had to be CO:COLLECTION_SUMMARY euthanized for humane reasons prior to reaching the predetermined time point CO:COLLECTION_SUMMARY were excluded from the analysis. Small intestinal content from mice was CO:COLLECTION_SUMMARY collected following a diet switch and maintenance for two days without infection CO:COLLECTION_SUMMARY (n=18, 6 per diet) prior to sacrifice, snap-frozen, and stored at -80°C. CO:SAMPLE_TYPE Feces CO:STORAGE_CONDITIONS -80℃ #TREATMENT TR:TREATMENT_SUMMARY For the aspartate-free diet experiments, Groups (n=5) of 6-7 weeks old CBA mice TR:TREATMENT_SUMMARY were fed control diet A20073101 (Research Diets) or aspartate-free diet TR:TREATMENT_SUMMARY A20073102 (Research Diets) beginning 48h before S. Tm infection and were kept on TR:TREATMENT_SUMMARY the diets throughout the experiment. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Frozen mouse intestinal content samples were lysed in 1000 µl ice-cold lysis SP:SAMPLEPREP_SUMMARY buffer (1:1:2, v:v:v, acetonitrile: methanol: ammonium bicarbonate 0.1M - pH SP:SAMPLEPREP_SUMMARY 8.0) and sonicated individually using a probe tip sonicator at 50% power (10 SP:SAMPLEPREP_SUMMARY pulses). Isotopically labeled standards (phenylalanine and biotin) were added to SP:SAMPLEPREP_SUMMARY each sample to determine sample process variability as previously described ). SP:SAMPLEPREP_SUMMARY Homogenized samples were normalized by weight to the smallest amount of tissue SP:SAMPLEPREP_SUMMARY sample such that each sample contained an equal amount of intestinal content SP:SAMPLEPREP_SUMMARY into a 200 µl final volume of lysate. Proteins were precipitated from SP:SAMPLEPREP_SUMMARY individual samples by the addition of 800 µL of ice-cold methanol followed by SP:SAMPLEPREP_SUMMARY overnight incubation at -80°C. Precipitated proteins were pelleted by SP:SAMPLEPREP_SUMMARY centrifugation (10k rpm, 10 min) and metabolite extracts were dried down in SP:SAMPLEPREP_SUMMARY vacuo and stored at -80°C. Samples were reconstituted in 100 µL H2O, and 100 SP:SAMPLEPREP_SUMMARY µL MeOH with vortex mixing after each addition. Samples were incubated at room SP:SAMPLEPREP_SUMMARY temperature for 10 min followed by liquid-liquid extraction. For liquid-liquid SP:SAMPLEPREP_SUMMARY extraction (LLE), 800 µL MTBE was added with vortex mixing for 30 sec followed SP:SAMPLEPREP_SUMMARY by incubation on ice for 10 min and centrifugation at 15k rpm for 15 min at 4 SP:SAMPLEPREP_SUMMARY °C. The lower (hydrophilic) fraction was transferred into a new Eppendorf tube, SP:SAMPLEPREP_SUMMARY dried in vacuo, and stored at -80 °C until further use. SP:PROCESSING_STORAGE_CONDITIONS -80℃ SP:EXTRACT_STORAGE -80℃ SP:SAMPLE_RESUSPENSION Prior to mass spectrometry analysis, individual hydrophilic extracts were SP:SAMPLE_RESUSPENSION reconstituted in 100 µL acetonitrile/water (80:20, v/v) containing isotopically SP:SAMPLE_RESUSPENSION labeled standards, tryptophan, inosine, valine, and carnitine, and centrifuged SP:SAMPLE_RESUSPENSION for 5 min at 10,000 rpm to remove insoluble material. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE HILIC CH:INSTRUMENT_NAME Thermo Vanquish CH:COLUMN_NAME Waters XBridge BEH Amide (100 x 2.1mm,2.5um) CH:SOLVENT_A 90% water, 10% acetonitrile, 5mM Ammonium Formate, 0.1%FA CH:SOLVENT_B 10% water, 90% acetonitrile, 5mM Ammonium Formate, 0.1%FA CH:FLOW_GRADIENT 30 min; 0-1 min 95%B, 1-12 min 95-45%B, 12-15 min 45%B, 15-26 min 45-95%B, 26-30 CH:FLOW_GRADIENT min 95%B CH:FLOW_RATE 0.20mL/min CH:COLUMN_TEMPERATURE 30 #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 Data analysis was performed as previously described . Spectral features MS:MS_COMMENTS (retention time, m/z pairs) were extracted from the data using Progenesis QI MS:MS_COMMENTS v.3.0 (Non-linear Dynamics, Waters Corporation, Milford, MA). MS:MS_RESULTS_FILE ST003796_AN006241_Results.txt UNITS:peak intensity Has m/z:Yes Has RT:Yes RT units:Minutes #END