#METABOLOMICS WORKBENCH yash_thsti_20250804_233537 DATATRACK_ID:6254 STUDY_ID:ST004099 ANALYSIS_ID:AN006795 PROJECT_ID:PR002575 VERSION 1 CREATED_ON August 5, 2025, 12:23 am #PROJECT PR:PROJECT_TITLE A genome-scale metabolic model for deciphering the host metabolic perturbations PR:PROJECT_TITLE during Mycobacterium tuberculosis infection PR:PROJECT_TYPE Metabolomics PR:PROJECT_SUMMARY Conventional tuberculosis (TB) research predominantly relies on forward-designed PR:PROJECT_SUMMARY experiments, such as studying host responses to Mycobacterium tuberculosis (Mtb) PR:PROJECT_SUMMARY gene knockouts. While informative, these approaches offer only a partial view of PR:PROJECT_SUMMARY host-pathogen interactions and often overlook the broader alterations in the PR:PROJECT_SUMMARY host microenvironment during infection. To address this limitation, we adopted a PR:PROJECT_SUMMARY backtracing strategy to retrospectively identify host metabolic modulators and PR:PROJECT_SUMMARY link them to specific Mtb virulence factors. Using RNA-seq data from PR:PROJECT_SUMMARY Mtb-infected mouse lung tissue, we integrated transcriptomic profiles into a PR:PROJECT_SUMMARY genome-scale metabolic model to predict host metabolic genes essential for Mtb PR:PROJECT_SUMMARY survival. This analysis identified 18 host proteins as putative modulators. We PR:PROJECT_SUMMARY then constructed a host-pathogen protein interaction network, which connected PR:PROJECT_SUMMARY these host factors to 9 Mtb proteins. Experimental validation using Mtb PR:PROJECT_SUMMARY knockdown strains confirmed that three genes Rv1970, Rv0243, and Rv2234 play key PR:PROJECT_SUMMARY roles in manipulating host responses rather than supporting intrinsic bacterial PR:PROJECT_SUMMARY viability. Further, we employed targeted proteomics and untargeted metabolomics PR:PROJECT_SUMMARY analyses on THP-1 macrophages infected with these KD strains to dissect the PR:PROJECT_SUMMARY host-pathogen interaction mechanisms in greater detail. These findings in PR:PROJECT_SUMMARY troduce a novel framework for decoding host-pathogen interactions and lay the PR:PROJECT_SUMMARY groundwork for future host-directed therapy (HDT) strategies targeting PR:PROJECT_SUMMARY Mtb-induced host vulnerabilities. PR:INSTITUTE Translational health science and technology institute PR:DEPARTMENT NCD PR:LABORATORY Biomarker lab PR:LAST_NAME Kumar PR:FIRST_NAME Yashwant PR:ADDRESS NCR Biotech Science Cluster,, Faridabad, Haryana, 121001, India PR:EMAIL y.kumar@thsti.res.in PR:PHONE 01292876496 PR:FUNDING_SOURCE THSTI #STUDY ST:STUDY_TITLE A genome-scale metabolic model for deciphering the host metabolic perturbations ST:STUDY_TITLE during Mycobacterium tuberculosis infection ST:STUDY_TYPE Metabolomics ST:STUDY_SUMMARY Conventional tuberculosis (TB) research predominantly relies on forward-designed ST:STUDY_SUMMARY experiments, such as studying host responses to Mycobacterium tuberculosis (Mtb) ST:STUDY_SUMMARY gene knockouts. While informative, these approaches offer only a partial view of ST:STUDY_SUMMARY host-pathogen interactions and often overlook the broader alterations in the ST:STUDY_SUMMARY host microenvironment during infection. To address this limitation, we adopted a ST:STUDY_SUMMARY backtracing strategy to retrospectively identify host metabolic modulators and ST:STUDY_SUMMARY link them to specific Mtb virulence factors. Using RNA-seq data from ST:STUDY_SUMMARY Mtb-infected mouse lung tissue, we integrated transcriptomic profiles into a ST:STUDY_SUMMARY genome-scale metabolic model to predict host metabolic genes essential for Mtb ST:STUDY_SUMMARY survival. This analysis identified 18 host proteins as putative modulators. We ST:STUDY_SUMMARY then constructed a host-pathogen protein interaction network, which connected ST:STUDY_SUMMARY these host factors to 9 Mtb proteins. Experimental validation using Mtb ST:STUDY_SUMMARY knockdown strains confirmed that three genes Rv1970, Rv0243, and Rv2234 play key ST:STUDY_SUMMARY roles in manipulating host responses rather than supporting intrinsic bacterial ST:STUDY_SUMMARY viability. Further, we employed targeted proteomics and untargeted metabolomics ST:STUDY_SUMMARY analyses on THP-1 macrophages infected with these KD strains to dissect the ST:STUDY_SUMMARY host-pathogen interaction mechanisms in greater detail. These findings in ST:STUDY_SUMMARY troduce a novel framework for decoding host-pathogen interactions and lay the ST:STUDY_SUMMARY groundwork for future host-directed therapy (HDT) strategies targeting ST:STUDY_SUMMARY Mtb-induced host vulnerabilities. ST:INSTITUTE Translational health science and technology institute ST:DEPARTMENT NCD ST:LABORATORY Biomarker lab ST:LAST_NAME Kumar ST:FIRST_NAME Yashwant ST:ADDRESS NCR Biotech Science Cluster,, Faridabad, Haryana, 121001, India ST:EMAIL y.kumar@thsti.res.in ST:PHONE +911292876496 #SUBJECT SU:SUBJECT_TYPE Cultured cells SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 #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 - 1 Treatment:Uninfected1 | Sample source:Human cell line RAW_FILE_NAME(Raw file)=1.mzXML SUBJECT_SAMPLE_FACTORS - 2 Treatment:Uninfected2 | Sample source:Human cell line RAW_FILE_NAME(Raw file)=2.mzXML SUBJECT_SAMPLE_FACTORS - 3 Treatment:Uninfected3 | Sample source:Human cell line RAW_FILE_NAME(Raw file)=3.mzXML SUBJECT_SAMPLE_FACTORS - 4 Treatment:Uninfected4 | Sample source:Human cell line RAW_FILE_NAME(Raw file)=4.mzXML SUBJECT_SAMPLE_FACTORS - 5 Treatment:Uninfected5 | Sample source:Human cell line RAW_FILE_NAME(Raw file)=5.mzXML SUBJECT_SAMPLE_FACTORS - 6 Treatment:Uninfected6 | Sample source:Human cell line RAW_FILE_NAME(Raw file)=6.mzXML SUBJECT_SAMPLE_FACTORS - 7 Treatment:Rv0410-Atc1 | Sample source:Human cell line RAW_FILE_NAME(Raw file)=7.mzXML SUBJECT_SAMPLE_FACTORS - 8 Treatment:Rv0410-Atc2 | Sample source:Human cell line RAW_FILE_NAME(Raw file)=8.mzXML SUBJECT_SAMPLE_FACTORS - 9 Treatment:Rv0410-Atc3 | Sample source:Human cell line RAW_FILE_NAME(Raw file)=9.mzXML SUBJECT_SAMPLE_FACTORS - 10 Treatment:Rv0410-Atc4 | Sample source:Human cell line RAW_FILE_NAME(Raw file)=10.mzXML SUBJECT_SAMPLE_FACTORS - 11 Treatment:Rv0410-Atc5 | Sample source:Human cell line RAW_FILE_NAME(Raw file)=11.mzXML SUBJECT_SAMPLE_FACTORS - 12 Treatment:Rv0410-Atc6 | Sample source:Human cell line RAW_FILE_NAME(Raw file)=12.mzXML SUBJECT_SAMPLE_FACTORS - 13 Treatment:Rv0410+Atc1 | Sample source:Human cell line RAW_FILE_NAME(Raw file)=13.mzXML SUBJECT_SAMPLE_FACTORS - 14 Treatment:Rv0410+Atc2 | Sample source:Human cell line RAW_FILE_NAME(Raw file)=14.mzXML SUBJECT_SAMPLE_FACTORS - 15 Treatment:Rv0410+Atc3 | Sample source:Human cell line RAW_FILE_NAME(Raw file)=15.mzXML SUBJECT_SAMPLE_FACTORS - 16 Treatment:Rv0410+Atc4 | Sample source:Human cell line RAW_FILE_NAME(Raw file)=16.mzXML SUBJECT_SAMPLE_FACTORS - 17 Treatment:Rv0410+Atc5 | Sample source:Human cell line RAW_FILE_NAME(Raw file)=17.mzXML SUBJECT_SAMPLE_FACTORS - 18 Treatment:Rv0410+Atc6 | Sample source:Human cell line RAW_FILE_NAME(Raw file)=18.mzXML SUBJECT_SAMPLE_FACTORS - 19 Treatment:Rv2234-Atc1 | Sample source:Human cell line RAW_FILE_NAME(Raw file)=19.mzXML SUBJECT_SAMPLE_FACTORS - 20 Treatment:Rv2234-Atc2 | Sample source:Human cell line RAW_FILE_NAME(Raw file)=20.mzXML SUBJECT_SAMPLE_FACTORS - 21 Treatment:Rv2234-Atc3 | Sample source:Human cell line RAW_FILE_NAME(Raw file)=21.mzXML SUBJECT_SAMPLE_FACTORS - 22 Treatment:Rv2234-Atc4 | Sample source:Human cell line RAW_FILE_NAME(Raw file)=22.mzXML SUBJECT_SAMPLE_FACTORS - 23 Treatment:Rv2234-Atc5 | Sample source:Human cell line RAW_FILE_NAME(Raw file)=23.mzXML SUBJECT_SAMPLE_FACTORS - 24 Treatment:Rv2234-Atc6 | Sample source:Human cell line RAW_FILE_NAME(Raw file)=24.mzXML SUBJECT_SAMPLE_FACTORS - 25 Treatment:Rv2234+Atc1 | Sample source:Human cell line RAW_FILE_NAME(Raw file)=25.mzXML SUBJECT_SAMPLE_FACTORS - 26 Treatment:Rv2234+Atc2 | Sample source:Human cell line RAW_FILE_NAME(Raw file)=26.mzXML SUBJECT_SAMPLE_FACTORS - 27 Treatment:Rv2234+Atc3 | Sample source:Human cell line RAW_FILE_NAME(Raw file)=27.mzXML SUBJECT_SAMPLE_FACTORS - 28 Treatment:Rv2234+Atc4 | Sample source:Human cell line RAW_FILE_NAME(Raw file)=28.mzXML SUBJECT_SAMPLE_FACTORS - 29 Treatment:Rv2234+Atc5 | Sample source:Human cell line RAW_FILE_NAME(Raw file)=29.mzXML SUBJECT_SAMPLE_FACTORS - 30 Treatment:Rv2234+Atc6 | Sample source:Human cell line RAW_FILE_NAME(Raw file)=30.mzXML SUBJECT_SAMPLE_FACTORS - 31 Treatment:Rv0243-Atc1 | Sample source:Human cell line RAW_FILE_NAME(Raw file)=31.mzXML SUBJECT_SAMPLE_FACTORS - 32 Treatment:Rv0243-Atc2 | Sample source:Human cell line RAW_FILE_NAME(Raw file)=32.mzXML SUBJECT_SAMPLE_FACTORS - 33 Treatment:Rv0243-Atc3 | Sample source:Human cell line RAW_FILE_NAME(Raw file)=33.mzXML SUBJECT_SAMPLE_FACTORS - 34 Treatment:Rv0243-Atc4 | Sample source:Human cell line RAW_FILE_NAME(Raw file)=34.mzXML SUBJECT_SAMPLE_FACTORS - 35 Treatment:Rv0243-Atc5 | Sample source:Human cell line RAW_FILE_NAME(Raw file)=35.mzXML SUBJECT_SAMPLE_FACTORS - 36 Treatment:Rv0243-Atc6 | Sample source:Human cell line RAW_FILE_NAME(Raw file)=36.mzXML SUBJECT_SAMPLE_FACTORS - 37 Treatment:Rv0243+Atc1 | Sample source:Human cell line RAW_FILE_NAME(Raw file)=37.mzXML SUBJECT_SAMPLE_FACTORS - 38 Treatment:Rv0243+Atc2 | Sample source:Human cell line RAW_FILE_NAME(Raw file)=38.mzXML SUBJECT_SAMPLE_FACTORS - 39 Treatment:Rv0243+Atc3 | Sample source:Human cell line RAW_FILE_NAME(Raw file)=39.mzXML SUBJECT_SAMPLE_FACTORS - 40 Treatment:Rv0243+Atc4 | Sample source:Human cell line RAW_FILE_NAME(Raw file)=40.mzXML SUBJECT_SAMPLE_FACTORS - 41 Treatment:Rv0243+Atc5 | Sample source:Human cell line RAW_FILE_NAME(Raw file)=41.mzXML SUBJECT_SAMPLE_FACTORS - 42 Treatment:Rv0243+Atc6 | Sample source:Human cell line RAW_FILE_NAME(Raw file)=42.mzXML SUBJECT_SAMPLE_FACTORS - 43 Treatment:Rv3774-Atc1 | Sample source:Human cell line RAW_FILE_NAME(Raw file)=43.mzXML SUBJECT_SAMPLE_FACTORS - 44 Treatment:Rv3774-Atc2 | Sample source:Human cell line RAW_FILE_NAME(Raw file)=44.mzXML SUBJECT_SAMPLE_FACTORS - 45 Treatment:Rv3774-Atc3 | Sample source:Human cell line RAW_FILE_NAME(Raw file)=45.mzXML SUBJECT_SAMPLE_FACTORS - 46 Treatment:Rv3774-Atc4 | Sample source:Human cell line RAW_FILE_NAME(Raw file)=46.mzXML SUBJECT_SAMPLE_FACTORS - 47 Treatment:Rv3774-Atc5 | Sample source:Human cell line RAW_FILE_NAME(Raw file)=47.mzXML SUBJECT_SAMPLE_FACTORS - 48 Treatment:Rv3774-Atc6 | Sample source:Human cell line RAW_FILE_NAME(Raw file)=48.mzXML SUBJECT_SAMPLE_FACTORS - 49 Treatment:Rv3774+Atc1 | Sample source:Human cell line RAW_FILE_NAME(Raw file)=49.mzXML SUBJECT_SAMPLE_FACTORS - 50 Treatment:Rv3774+Atc2 | Sample source:Human cell line RAW_FILE_NAME(Raw file)=50.mzXML SUBJECT_SAMPLE_FACTORS - 51 Treatment:Rv3774+Atc3 | Sample source:Human cell line RAW_FILE_NAME(Raw file)=51.mzXML SUBJECT_SAMPLE_FACTORS - 52 Treatment:Rv3774+Atc4 | Sample source:Human cell line RAW_FILE_NAME(Raw file)=52.mzXML SUBJECT_SAMPLE_FACTORS - 53 Treatment:Rv3774+Atc5 | Sample source:Human cell line RAW_FILE_NAME(Raw file)=53.mzXML SUBJECT_SAMPLE_FACTORS - 54 Treatment:Rv3774+Atc6 | Sample source:Human cell line RAW_FILE_NAME(Raw file)=54.mzXML SUBJECT_SAMPLE_FACTORS - 55 Treatment:Rv1970-Atc1 | Sample source:Human cell line RAW_FILE_NAME(Raw file)=55.mzXML SUBJECT_SAMPLE_FACTORS - 56 Treatment:Rv1970-Atc2 | Sample source:Human cell line RAW_FILE_NAME(Raw file)=56.mzXML SUBJECT_SAMPLE_FACTORS - 57 Treatment:Rv1970-Atc3 | Sample source:Human cell line RAW_FILE_NAME(Raw file)=57.mzXML SUBJECT_SAMPLE_FACTORS - 58 Treatment:Rv1970-Atc4 | Sample source:Human cell line RAW_FILE_NAME(Raw file)=58.mzXML SUBJECT_SAMPLE_FACTORS - 59 Treatment:Rv1970-Atc5 | Sample source:Human cell line RAW_FILE_NAME(Raw file)=59.mzXML SUBJECT_SAMPLE_FACTORS - 60 Treatment:Rv1970-Atc6 | Sample source:Human cell line RAW_FILE_NAME(Raw file)=60.mzXML SUBJECT_SAMPLE_FACTORS - 61 Treatment:Rv1970+Atc1 | Sample source:Human cell line RAW_FILE_NAME(Raw file)=61.mzXML SUBJECT_SAMPLE_FACTORS - 62 Treatment:Rv1970+Atc2 | Sample source:Human cell line RAW_FILE_NAME(Raw file)=62.mzXML SUBJECT_SAMPLE_FACTORS - 63 Treatment:Rv1970+Atc3 | Sample source:Human cell line RAW_FILE_NAME(Raw file)=63.mzXML SUBJECT_SAMPLE_FACTORS - 64 Treatment:Rv1970+Atc4 | Sample source:Human cell line RAW_FILE_NAME(Raw file)=64.mzXML SUBJECT_SAMPLE_FACTORS - 65 Treatment:Rv1970+Atc5 | Sample source:Human cell line RAW_FILE_NAME(Raw file)=65.mzXML SUBJECT_SAMPLE_FACTORS - 66 Treatment:Rv1970+Atc6 | Sample source:Human cell line RAW_FILE_NAME(Raw file)=66.mzXML #COLLECTION CO:COLLECTION_SUMMARY THP-1 cells (2x10 6 ) were seeded in 6-well plates and treated with 50 ng/mL PMA CO:COLLECTION_SUMMARY for 36 hours to induce differentiation. After PMA treatment, the medium was CO:COLLECTION_SUMMARY replaced with fresh RPMI, and the cells were allowed to mature for an additional CO:COLLECTION_SUMMARY 24 hours. Mtb infection was performed at a MOI of 5 and un-infected cells were CO:COLLECTION_SUMMARY maintained in parallel culture as control. After 4 hours of infection, CO:COLLECTION_SUMMARY extracellular bacteria were removed by three washes with pre-warmed PBS. A CO:COLLECTION_SUMMARY complete RPMI medium supplemented with 400 ng/mL ATc was added to the wells to CO:COLLECTION_SUMMARY induce the knockdown effect. The infected cells were incubated for 48 hours CO:COLLECTION_SUMMARY before being washed again with pre-warmed PBS. CO:SAMPLE_TYPE Blood (whole) #TREATMENT TR:TREATMENT_SUMMARY THP-1 cells (2x10 6 ) were seeded in 6-well plates and treated with 50 ng/mL PMA TR:TREATMENT_SUMMARY for 36 hours to induce differentiation. After PMA treatment, the medium was TR:TREATMENT_SUMMARY replaced with fresh RPMI, and the cells were allowed to mature for an additional TR:TREATMENT_SUMMARY 24 hours. Mtb infection was performed at a MOI of 5 and un-infected cells were TR:TREATMENT_SUMMARY maintained in parallel culture as control. After 4 hours of infection, TR:TREATMENT_SUMMARY extracellular bacteria were removed by three washes with pre-warmed PBS. A TR:TREATMENT_SUMMARY complete RPMI medium supplemented with 400 ng/mL ATc was added to the wells to TR:TREATMENT_SUMMARY induce the knockdown effect. The infected cells were incubated for 48 hours TR:TREATMENT_SUMMARY before being washed again with pre-warmed PBS. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY To quench metabolism and extract metabolites, 1 mL of chilled methanol (HPLC SP:SAMPLEPREP_SUMMARY grade; Sigma) was added to the cells. The cells were harvested on ice using a SP:SAMPLEPREP_SUMMARY scraper, and the resulting extracts were transferred to pre-cooled SP:SAMPLEPREP_SUMMARY microcentrifuge tubes (MCTs) maintained at 4°C. The extracts were vortexed for SP:SAMPLEPREP_SUMMARY 15 minutes and centrifuged at 12000 rpm for 10 minutes at 4°C. After SP:SAMPLEPREP_SUMMARY centrifugation, the supernatants were transferred to fresh pre-cooled MCTs SP:SAMPLEPREP_SUMMARY (4°C), dried under vacuum, and reconstituted. The reconstituted samples were SP:SAMPLEPREP_SUMMARY stored at -80°C until further analysis by liquid chromatography-mass SP:SAMPLEPREP_SUMMARY spectrometry (LC-MS). #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Extracted metabolites were separated on UPLC ultimate 3,000. Data were acquired CH:CHROMATOGRAPHY_SUMMARY on the reverse phase and HILIC column with positive and negative ionization CH:CHROMATOGRAPHY_SUMMARY modes. The reverse phase column was HSS T3, and the HILIC column was XBridge BEH CH:CHROMATOGRAPHY_SUMMARY Amide (Waters Corporation). CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Thermo Dionex Ultimate 3000 RS CH:COLUMN_NAME Waters ACQUITY UPLC HSS T3 (100 x 2.1mm,1.8um) CH:SOLVENT_A water+0.1% FA CH:SOLVENT_B Methanol+0.1% FA CH:FLOW_GRADIENT 1% B to 95% B in 10 minutes CH:FLOW_RATE 300 ul/min CH:COLUMN_TEMPERATURE 40°C #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Fusion Tribrid Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS An Orbitrap Fusion mass spectrometer (Thermo Scientific), coupled with a heated MS:MS_COMMENTS electrospray ion source, was used for data acquisition. For MS1 mode, a mass MS:MS_COMMENTS resolution was kept at 120,000, and for MS2 acquisition, a mass resolution was MS:MS_COMMENTS 30,000. The mass range of data acquisition was 60–900 Da. Extracted MS:MS_COMMENTS metabolites were separated on UPLC ultimate 3,000. Data were acquired on the MS:MS_COMMENTS reverse phase and HILIC column with positive and negative ionization modes. The MS:MS_COMMENTS reverse phase column was HSS T3, and the HILIC column was XBridge BEH Amide MS:MS_COMMENTS (Waters Corporation). For polar compound separation, solvent A was 20 mM MS:MS_COMMENTS ammonium acetate in water of PH 9.0, and mobile phase B was 100% acetonitrile. MS:MS_COMMENTS The elution gradient starts from 85% B to 10% B over 14 min with a flow rate of MS:MS_COMMENTS 0.35 ml/min. For the reverse phase, Solvent A was water, and B was methanol with MS:MS_COMMENTS 0.1% formic acid added in both. The elution gradient starts with 1% B to 95% B MS:MS_COMMENTS over 10 min with a flow rate of 0.3 ml/min. sample injection volume was 5 µl. MS:MS_RESULTS_FILE ST004099_AN006795_Results.txt UNITS:relative intensity Has m/z:Yes Has RT:Yes RT units:Seconds #END