Understanding metabolic adaptations of Mycobacterium tuberculosis in Alveolar epithelial cells during infection
Description
Isotopic labelling with 13C-labelled proteinogenic amino acids was carried out in A549 cells infected with a range of Mycobacterium tuberculosis strains, including both virulent and avirulent laboratory strains, as well as drug-sensitive and drug-resistant clinical isolates, across various time points. The study also included controls with heat-killed H37Rv-infected cells, uninfected cells, and unlabelled (12C) samples for reference. 12C labelled samples - set a, 13C labelled samples - set b The Objective -1 folder contains the raw data files for 105 samples (6 time points x 5 replicates x 3 groups) + (3 time points x 5 replicates x 1 group), excluding the blank folder. The Objective -2 folder contains the raw data files for 90 samples (3 time points x 5 replicates x 6 groups), excluding the blank folder.
Files
Steps to reproduce
A549 cells were cultured in DMEM/F12 with 10% FBS and sodium bicarbonate at 37 °C and 5% CO2. For ^13C glucose labelling, cells were grown in SILAC Advanced DMEM/F-12 with 5% dialyzed FBS, U-^13C6 or unlabelled glucose. Mycobacterium tuberculosis (Mtb) strains (H37Rv, H37Ra, S4, S5, S6, and S11) were grown in Middlebrook 7H9 broth with glycerol, Tween-80, and OADC, and harvested at OD600nm = 0.6. For infection, A549 cells were seeded, infected with Mtb, and incubated. At 8 hours post-infection, the media was replaced with fresh media containing U-12C6 or U-13C6 glucose. Cell culture media was collected, filtered, and stored at -80 ºC. Cells were washed, quenched with methanol and water, and extracted with chloroform. Phases were separated, and proteins were acid-hydrolyzed, vacuum-dried, and derivatized with TBDMS for GC-MS analysis. GC-MS data was acquired using a 7890 Gas Chromatograph and a 5975C quadrupole mass spectrometer (Agilent Technologies). All 450 samples were analyzed in a single batch, processed in groups of 20 with 2 QC samples at the start and end. Derivatized samples (1 μL) were injected into a Restek RTX-5 column in splitless mode. Helium was used as the carrier gas at 1.3 mL/min, with a solvent delay of 6 min. The oven program began at 120 °C, ramped to 270 °C at 4°C/min, then to 320 °C at 20 °C/min. The ion source temperature was 230 °C, the quadrupole temperature was 150 °C, and data were acquired from 50 to 600 m/z over 49 minutes using electron ionization (70 eV). Data acquisition was controlled by Agilent ChemStation software and completed within 24-48 hours of derivatization. Sample acquisition was completed within 24-48 hours post-derivatization. GC-MS spectra were baseline-corrected with MetAlign software to determine mass isotopologue distributions (MIDs). Peaks were identified using ChemStation software and compared with the NIST17 Library and commercial standards. MIDs were corrected for natural isotope abundance using Isocor software, with derivatization formulas manually calculated. Final corrected MIDs were used to compute average ^13C abundance. Amino acid fragments from unlabelled samples with 13C ≤ 2% were considered valid.