Study Data
| Project uploaded by: | Yashwant |
| Project ID: | IMP_100052 |
| Title: | A genome-scale metabolic model for deciphering the host metabolic perturbations during Mycobacterium tuberculosis infection |
| Project Description: | Conventional tuberculosis (TB) research predominantly relies on forward-designed experiments, such as studying host responses to Mycobacterium tuberculosis (Mtb) gene knockouts. While informative, these approaches offer only a partial view of host-pathogen interactions and often overlook the broader alterations in the host microenvironment during infection. To address this limitation, we adopted a backtracing strategy to retrospectively identify host metabolic modulators and link them to specific Mtb virulence factors. Using RNA-seq data from Mtb-infected mouse lung tissue, we integrated transcriptomic profiles into a genome-scale metabolic model to predict host metabolic genes essential for Mtb survival. This analysis identified 18 host proteins as putative modulators. We then constructed a host-pathogen protein interaction network, which connected these host factors to 9 Mtb proteins. Experimental validation using Mtb knockdown strains confirmed that three genes Rv1970, Rv0243, and Rv2234 play key roles in manipulating host responses rather than supporting intrinsic bacterial viability. Further, we employed targeted proteomics and untargeted metabolomics analyses on THP-1 macrophages infected with these KD strains to dissect the host-pathogen interaction mechanisms in greater detail. These findings in troduce a novel framework for decoding host-pathogen interactions and lay the groundwork for future host-directed therapy (HDT) strategies targeting Mtb-induced host vulnerabilities. |
| Research Area: | Biological Sciences |
| Funding Source: | Translational Research Program (TRP) (No. BT/PR30159/MED/15/188/2018) of Department of Biotechnology (DBT), Govt. of India. |
| Project Contributors: | Yashwant Kumar |
| Sr.No | Sample ID | Sample Name | Organism | Source | Sample Preparation Protocol | Sample Type | Experimental Condition | Time of treatment | Variant/Variety | Gender | Age | Replicates | Storage Conditions | Extraction Protocol | Number of files per sample |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 61 | IMSM_103729 | Uninfected1 | Homo sapiens | 9606 | Human acute monocytic leukemia cell line | THP-1 cells (2x10^6) were seeded in 6-well plates and treated with 50 ng/mL PMA for 36 hours to induce differentiation. After PMA treatment, the medium was replaced with fresh RPMI, and the cells were allowed to mature for an additional 24 hours. Mtb infection was performed at a MOI of 5 and un-infected cells were maintained in parallel culture as control. After 4 hours of infection, extracellular bacteria were removed by three washes with pre-warmed PBS. A complete RPMI medium supplemented with 400 ng/mL ATc was added to the wells to induce the knockdown effect. The infected cells were incubated for 48 hours before being washed again with pre-warmed PBS. | Human THP-1 Macrophages | Uninfected | 48 hours | NA | NA | NA | Yes | NA |
To quench metabolism and extract metabolites, 1 mL of chilled methanol (HPLC grade; Sigma) was added to the cells. The cells were harvested on ice using a scraper, and the resulting extracts were transferred to pre-cooled microcentrifuge tubes (MCTs) maintained at 4°C. The extracts were vortexed for 15 minutes and centrifuged at 12000 rpm for 10 minutes at 4°C. After centrifugation, the supernatants were transferred to fresh pre-cooled MCTs (4°C), dried under vacuum, and reconstituted. The reconstituted samples were stored at -80°C until further analysis by liquid chromatography-mass spectrometry (LC-MS). |
4 |
| 62 | IMSM_103730 | Uninfected2 | Homo sapiens | 9606 | Human acute monocytic leukemia cell line | THP-1 cells (2x10^6) were seeded in 6-well plates and treated with 50 ng/mL PMA for 36 hours to induce differentiation. After PMA treatment, the medium was replaced with fresh RPMI, and the cells were allowed to mature for an additional 24 hours. Mtb infection was performed at a MOI of 5 and un-infected cells were maintained in parallel culture as control. After 4 hours of infection, extracellular bacteria were removed by three washes with pre-warmed PBS. A complete RPMI medium supplemented with 400 ng/mL ATc was added to the wells to induce the knockdown effect. The infected cells were incubated for 48 hours before being washed again with pre-warmed PBS. | Human THP-1 Macrophages | Uninfected | 48 hours | NA | NA | NA | Yes | NA |
To quench metabolism and extract metabolites, 1 mL of chilled methanol (HPLC grade; Sigma) was added to the cells. The cells were harvested on ice using a scraper, and the resulting extracts were transferred to pre-cooled microcentrifuge tubes (MCTs) maintained at 4°C. The extracts were vortexed for 15 minutes and centrifuged at 12000 rpm for 10 minutes at 4°C. After centrifugation, the supernatants were transferred to fresh pre-cooled MCTs (4°C), dried under vacuum, and reconstituted. The reconstituted samples were stored at -80°C until further analysis by liquid chromatography-mass spectrometry (LC-MS). |
4 |
| 63 | IMSM_103731 | Uninfected3 | Homo sapiens | 9606 | Human acute monocytic leukemia cell line | THP-1 cells (2x10^6) were seeded in 6-well plates and treated with 50 ng/mL PMA for 36 hours to induce differentiation. After PMA treatment, the medium was replaced with fresh RPMI, and the cells were allowed to mature for an additional 24 hours. Mtb infection was performed at a MOI of 5 and un-infected cells were maintained in parallel culture as control. After 4 hours of infection, extracellular bacteria were removed by three washes with pre-warmed PBS. A complete RPMI medium supplemented with 400 ng/mL ATc was added to the wells to induce the knockdown effect. The infected cells were incubated for 48 hours before being washed again with pre-warmed PBS. | Human THP-1 Macrophages | Uninfected | 48 hours | NA | NA | NA | Yes | NA |
To quench metabolism and extract metabolites, 1 mL of chilled methanol (HPLC grade; Sigma) was added to the cells. The cells were harvested on ice using a scraper, and the resulting extracts were transferred to pre-cooled microcentrifuge tubes (MCTs) maintained at 4°C. The extracts were vortexed for 15 minutes and centrifuged at 12000 rpm for 10 minutes at 4°C. After centrifugation, the supernatants were transferred to fresh pre-cooled MCTs (4°C), dried under vacuum, and reconstituted. The reconstituted samples were stored at -80°C until further analysis by liquid chromatography-mass spectrometry (LC-MS). |
4 |
| 64 | IMSM_103732 | Uninfected4 | Homo sapiens | 9606 | Human acute monocytic leukemia cell line | THP-1 cells (2x10^6) were seeded in 6-well plates and treated with 50 ng/mL PMA for 36 hours to induce differentiation. After PMA treatment, the medium was replaced with fresh RPMI, and the cells were allowed to mature for an additional 24 hours. Mtb infection was performed at a MOI of 5 and un-infected cells were maintained in parallel culture as control. After 4 hours of infection, extracellular bacteria were removed by three washes with pre-warmed PBS. A complete RPMI medium supplemented with 400 ng/mL ATc was added to the wells to induce the knockdown effect. The infected cells were incubated for 48 hours before being washed again with pre-warmed PBS. | Human THP-1 Macrophages | Uninfected | 48 hours | NA | NA | NA | Yes | NA |
To quench metabolism and extract metabolites, 1 mL of chilled methanol (HPLC grade; Sigma) was added to the cells. The cells were harvested on ice using a scraper, and the resulting extracts were transferred to pre-cooled microcentrifuge tubes (MCTs) maintained at 4°C. The extracts were vortexed for 15 minutes and centrifuged at 12000 rpm for 10 minutes at 4°C. After centrifugation, the supernatants were transferred to fresh pre-cooled MCTs (4°C), dried under vacuum, and reconstituted. The reconstituted samples were stored at -80°C until further analysis by liquid chromatography-mass spectrometry (LC-MS). |
4 |
| 65 | IMSM_103733 | Uninfected5 | Homo sapiens | 9606 | Human acute monocytic leukemia cell line | THP-1 cells (2x10^6) were seeded in 6-well plates and treated with 50 ng/mL PMA for 36 hours to induce differentiation. After PMA treatment, the medium was replaced with fresh RPMI, and the cells were allowed to mature for an additional 24 hours. Mtb infection was performed at a MOI of 5 and un-infected cells were maintained in parallel culture as control. After 4 hours of infection, extracellular bacteria were removed by three washes with pre-warmed PBS. A complete RPMI medium supplemented with 400 ng/mL ATc was added to the wells to induce the knockdown effect. The infected cells were incubated for 48 hours before being washed again with pre-warmed PBS. | Human THP-1 Macrophages | Uninfected | 48 hours | NA | NA | NA | Yes | NA |
To quench metabolism and extract metabolites, 1 mL of chilled methanol (HPLC grade; Sigma) was added to the cells. The cells were harvested on ice using a scraper, and the resulting extracts were transferred to pre-cooled microcentrifuge tubes (MCTs) maintained at 4°C. The extracts were vortexed for 15 minutes and centrifuged at 12000 rpm for 10 minutes at 4°C. After centrifugation, the supernatants were transferred to fresh pre-cooled MCTs (4°C), dried under vacuum, and reconstituted. The reconstituted samples were stored at -80°C until further analysis by liquid chromatography-mass spectrometry (LC-MS). |
4 |
| 66 | IMSM_103734 | Uninfected6 | Homo sapiens | 9606 | Human acute monocytic leukemia cell line | THP-1 cells (2x10^6) were seeded in 6-well plates and treated with 50 ng/mL PMA for 36 hours to induce differentiation. After PMA treatment, the medium was replaced with fresh RPMI, and the cells were allowed to mature for an additional 24 hours. Mtb infection was performed at a MOI of 5 and un-infected cells were maintained in parallel culture as control. After 4 hours of infection, extracellular bacteria were removed by three washes with pre-warmed PBS. A complete RPMI medium supplemented with 400 ng/mL ATc was added to the wells to induce the knockdown effect. The infected cells were incubated for 48 hours before being washed again with pre-warmed PBS. | Human THP-1 Macrophages | Uninfected | 48 hours | NA | NA | NA | Yes | NA |
To quench metabolism and extract metabolites, 1 mL of chilled methanol (HPLC grade; Sigma) was added to the cells. The cells were harvested on ice using a scraper, and the resulting extracts were transferred to pre-cooled microcentrifuge tubes (MCTs) maintained at 4°C. The extracts were vortexed for 15 minutes and centrifuged at 12000 rpm for 10 minutes at 4°C. After centrifugation, the supernatants were transferred to fresh pre-cooled MCTs (4°C), dried under vacuum, and reconstituted. The reconstituted samples were stored at -80°C until further analysis by liquid chromatography-mass spectrometry (LC-MS). |
4 |
| Sr.No | MS Exp ID | Sample Name/ID | Mass Spectrometer Type | MS Instrument Name | MS Instrument type | MS Ionization Method | Ion Mode/Scan Polarity | Data Transformation (Software/s Used) |
|---|---|---|---|---|---|---|---|---|
| 261 | IME_101968 | Uninfected3 / IMSM_103731 | LCMS (Liquid Chromatography- Mass Spectrometry) | Thermo Fusion Tribrid Orbitrap | Orbitrap | Electrospray Ionization - ESI | POSITIVE | NA |
| 262 | IME_101969 | Uninfected4 / IMSM_103732 | LCMS (Liquid Chromatography- Mass Spectrometry) | Thermo Fusion Tribrid Orbitrap | Orbitrap | Electrospray Ionization - ESI | POSITIVE | NA |
| 263 | IME_101970 | Uninfected5 / IMSM_103733 | LCMS (Liquid Chromatography- Mass Spectrometry) | Thermo Fusion Tribrid Orbitrap | Orbitrap | Electrospray Ionization - ESI | POSITIVE | NA |
| 264 | IME_101971 | Uninfected6 / IMSM_103734 | LCMS (Liquid Chromatography- Mass Spectrometry) | Thermo Fusion Tribrid Orbitrap | Orbitrap | Electrospray Ionization - ESI | POSITIVE | NA |
| Sr.No | First name | Last name | Organization | Designation | |
|---|---|---|---|---|---|
| 1 | Yashwant | Kumar | y.kumar@thsti.res.in | Translational Health Science And Technology Institute (THSTI) | principal_investigator |
| Sr.No | ftprun ID | MS Exp ID | MS Data Files |
|---|---|---|---|
| 11 | IMR_102322 | IME_101718 | HILIC_NEG_Rv0243+Atc6.mzXML |
| 12 | IMR_102323 | IME_101719 | HILIC_NEG_Rv0243-Atc6.mzXML |
| 13 | IMR_102324 | IME_101720 | HILIC_NEG_Rv0410+Atc1.mzXML |
| 14 | IMR_102325 | IME_101721 | HILIC_NEG_Rv0410-Atc1.mzXML |
| 15 | IMR_102326 | IME_101722 | HILIC_NEG_Rv0410+Atc2.mzXML |
| 16 | IMR_102327 | IME_101723 | HILIC_NEG_Rv0410-Atc2.mzXML |
| 17 | IMR_102328 | IME_101724 | HILIC_NEG_Rv0410+Atc3.mzXML |
| 18 | IMR_102329 | IME_101725 | HILIC_NEG_Rv0410-Atc3.mzXML |
| 19 | IMR_102330 | IME_101726 | HILIC_NEG_Rv0410+Atc4.mzXML |
| 20 | IMR_102331 | IME_101727 | HILIC_NEG_Rv0410-Atc4.mzXML |