#METABOLOMICS WORKBENCH sysu604_20230914_235109 DATATRACK_ID:4318 STUDY_ID:ST002877 ANALYSIS_ID:AN004714 VERSION 1 CREATED_ON 10-04-2023 #PROJECT PR:PROJECT_TITLE Exogenous L-Alanine promotes phagocytosis via dual regulations of TLR4 to PR:PROJECT_TITLE eliminate multidrug-resistant bacterial pathogens PR:PROJECT_TYPE MS quantitative analysis PR:PROJECT_SUMMARY Multidrug-resistant bacteria present a major threat to public health. Therefore, PR:PROJECT_SUMMARY new drugs or approaches are urgently needed to manage and mitigate this threat. PR:PROJECT_SUMMARY Here, we screen the molecular candidates that allow the survival of mice upon PR:PROJECT_SUMMARY multidrug-resistant Vibrio parahaemolyticus infection by integrated proteomic PR:PROJECT_SUMMARY and metabolomics analysis, where L-Alanine metabolism and phagocytosis are PR:PROJECT_SUMMARY highly correlated. The role of L-Alanine on boosting mouse survival is further PR:PROJECT_SUMMARY confirmed with in vivo bacterial challenge studies on multidrug-resistant PR:PROJECT_SUMMARY bacteria including V. parahaemolyticus, Escherichia coli, Pseudomonas PR:PROJECT_SUMMARY aeruginosa, Klebsiella pneumoniae. Functional studies demonstrate that exogenous PR:PROJECT_SUMMARY L-Alanine promotes phagocytosis to these different species of PR:PROJECT_SUMMARY multidrug-resistant pathogens. The underlying mechanism involves two events that PR:PROJECT_SUMMARY are L-Alanine-dependently increased TLR4 expression, and L-Alanine-enhanced TLR4 PR:PROJECT_SUMMARY signaling via increasing the biosynthesis and secretion of fatty acids such as PR:PROJECT_SUMMARY palmitate. Palmitate enhances the binding of LPS to TLR4 and thereby promotes PR:PROJECT_SUMMARY TLR4 dimmer formation and endocytosis for the subsequent activation of PI3K/Akt PR:PROJECT_SUMMARY and NF-κB pathways and phagocytosis of bacteria. These results suggest that PR:PROJECT_SUMMARY modulation of metabolic environment is a plausible approach for combating PR:PROJECT_SUMMARY infection with multidrug-resistant bacteria. PR:INSTITUTE sun yat-sen university PR:LAST_NAME jiang PR:FIRST_NAME ming PR:ADDRESS No. 135, Xingang Xi Road, Guangzhou, 510275, P. R. China, guangzhou, guangdong, PR:ADDRESS 510006, China PR:EMAIL jiangm28@mail.sysu.edu.cn PR:PHONE 13434283781 PR:DOI http://dx.doi.org/10.21228/M85M79 #STUDY ST:STUDY_TITLE Metabolic Profiling of Raw264.7 Mouse Macrophage Cells Cultured with Alanine ST:STUDY_SUMMARY To identify the catabolites of L-Alanine on promoting phagocytosis, GC-MS based ST:STUDY_SUMMARY metabolomics analysis was adopted to explore L-Alanine-reprogrammed metabolome. ST:STUDY_SUMMARY The metabolic flow of the TCA cycle was dysregulated. Meanwhile, six metabolites ST:STUDY_SUMMARY (oleate, palmitate, stearate, myristate, arachidonate and linoleate) in ST:STUDY_SUMMARY biosynthesis of saturated and unsaturated fatty acids were increased upon ST:STUDY_SUMMARY L-Alanine treatment, where palmitate was the biggest absolute increment in ST:STUDY_SUMMARY abundance. Thus, L-Alanine promotes the biosynthesis of fatty acids. ST:INSTITUTE Sun Yat-sen University ST:LAST_NAME jiang ST:FIRST_NAME ming ST:ADDRESS No. 135, Xingang Xi Road, Guangzhou, 510275, P. R. China, guangzhou, guangdong, ST:ADDRESS 510006, China ST:EMAIL jiangm28@mail.sysu.edu.cn ST:PHONE 13434283781 ST:SUBMIT_DATE 2023-09-14 #SUBJECT SU:SUBJECT_TYPE Cultured cells SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS - Ala-40-1-1 factor:Ala other=Wild-type; RAW_FILE_NAME=Ala-40-1-1.raw; RAW_FILE_NAME=- SUBJECT_SAMPLE_FACTORS - Ala-40-1-2 factor:Ala other=Wild-type; RAW_FILE_NAME=Ala-40-1-2.raw; RAW_FILE_NAME=- SUBJECT_SAMPLE_FACTORS - Ala-40-2-1 factor:Ala other=Wild-type; RAW_FILE_NAME=Ala-40-2-1.raw; RAW_FILE_NAME=- SUBJECT_SAMPLE_FACTORS - Ala-40-2-2 factor:Ala other=Wild-type; RAW_FILE_NAME=Ala-40-2-2.raw; RAW_FILE_NAME=- SUBJECT_SAMPLE_FACTORS - Ala-40-3-1 factor:Ala other=Wild-type; RAW_FILE_NAME=Ala-40-3-1.raw; RAW_FILE_NAME=- SUBJECT_SAMPLE_FACTORS - Ala-40-3-2 factor:Ala other=Wild-type; RAW_FILE_NAME=Ala-40-3-2.raw; RAW_FILE_NAME=- SUBJECT_SAMPLE_FACTORS - control-1-1 factor:Control other=Wild-type; RAW_FILE_NAME=control-1-1.raw; RAW_FILE_NAME=- SUBJECT_SAMPLE_FACTORS - control-1-2 factor:Control other=Wild-type; RAW_FILE_NAME=control-1-2.raw; RAW_FILE_NAME=- SUBJECT_SAMPLE_FACTORS - control-2-1 factor:Control other=Wild-type; RAW_FILE_NAME=control-2-1.raw; RAW_FILE_NAME=- SUBJECT_SAMPLE_FACTORS - control-2-2 factor:Control other=Wild-type; RAW_FILE_NAME=control-2-2.raw; RAW_FILE_NAME=- SUBJECT_SAMPLE_FACTORS - control-3-1 factor:Control other=Wild-type; RAW_FILE_NAME=control-3-1.raw; RAW_FILE_NAME=- SUBJECT_SAMPLE_FACTORS - control-3-2 factor:Control other=Wild-type; RAW_FILE_NAME=control-3-2.raw; RAW_FILE_NAME=- #COLLECTION CO:COLLECTION_SUMMARY Cells were counted, washed with cold PBS and then flash-frozen in liquid N2 CO:SAMPLE_TYPE Cultured cells #TREATMENT TR:TREATMENT_SUMMARY To trace L-Alanine metabolism, RAW264.7 cell were grown in DMEM (Hyclone) TR:TREATMENT_SUMMARY supplemented with 10% (v/v) cosmic calf (Hyclone), then transferred into TR:TREATMENT_SUMMARY L-Alanine-free medium and deprived of serum overnight. Subsequently, cells were TR:TREATMENT_SUMMARY incubated with 5 mM L-Alanine and 5mM [U-13C]-L-Alanine in serum-starved medium TR:TREATMENT_SUMMARY (DMEM/0.5% serum). Additionally, fresh media containing L-Alanine and TR:TREATMENT_SUMMARY [U-13C]-L-Alanine were exchanged 2 h before metabolite extraction for metabolic TR:TREATMENT_SUMMARY analysis. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Cells were homogenized with the first solvent (the mixture of chloroform, SP:SAMPLEPREP_SUMMARY methanol and water (1:2:1, v/v/v)) for 30 s at 4 °C and then centrifuged at SP:SAMPLEPREP_SUMMARY 12,000 rpm for 10 min at 4 °C. The supernatant was collected and deposit was SP:SAMPLEPREP_SUMMARY re-homogenized with the second solvent (methanol alone) before a second SP:SAMPLEPREP_SUMMARY centrifugation. The 2 supernatants were mixed, and aliquot of sample was SP:SAMPLEPREP_SUMMARY transferred to a GC sampling vial containing 5 μL 0.1 mg/mL ribitol (Sigma) as SP:SAMPLEPREP_SUMMARY an analytical internal standard and then dried in a vacuum centrifuge SP:SAMPLEPREP_SUMMARY concentrator before the subsequent derivatization. A total of 2 technical SP:SAMPLEPREP_SUMMARY replicates were prepared for each sample. #CHROMATOGRAPHY CH:INSTRUMENT_NAME Thermo Scientific Trace GC Ultra with DSQ II GC/MS CH:COLUMN_NAME Agilent DB5-MS (30m x 0.25mm, 0.25um) CH:COLUMN_TEMPERATURE 270 °C CH:FLOW_GRADIENT none CH:FLOW_RATE 1.0 mL/min CH:SOLVENT_A none CH:SOLVENT_B none CH:CHROMATOGRAPHY_TYPE GC #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Scientific Trace GC Ultra with DSQ II GC/MS MS:INSTRUMENT_TYPE Triple quadrupole MS:MS_TYPE EI MS:MS_COMMENTS samples was derivatized and then used to firstly protect carbonyl moieties MS:MS_COMMENTS through methoximation, through a 90 min 37 ℃ reaction with 40 μL of 20 mg/mL MS:MS_COMMENTS methoxyamine hydrochloride (Sigma-Aldrich) in pyridine, followed by MS:MS_COMMENTS derivatization of acidic protons through a 30 min 37 0C reaction with the MS:MS_COMMENTS addition of 80 μL N-methyl-N-trimethylsilyltrifluoroace-tamide (MSTFA, MS:MS_COMMENTS Sigma-Aldrich). The derivatized sample of 1 μL was injected into a 30m × 250 MS:MS_COMMENTS μm i.d. × 0.25 μm DBS-MS column using splitless injection and analysis was MS:MS_COMMENTS carried out by Trace DSQ II (Thermo Scientific). The initial temperature of the MS:MS_COMMENTS GC oven was held at 85 0C for 5 min followed by an increase to 330 0C at a rate MS:MS_COMMENTS of 15 0C min-1 then held for 5 min. Helium was used as carrier gas and flow was MS:MS_COMMENTS kept constant at 1 mL min-1. The MS was operated in a range of 50-600 m/z. MS:ION_MODE POSITIVE MS:MS_RESULTS_FILE ST002877_AN004714_Results.txt UNITS:Peak area Has m/z:Yes Has RT:Yes RT units:Minutes #END