Host-dependent resistance of Group A Streptococcus to sulfamethoxazole mediated by a horizontally-acquired reduced folate transporter - MS and NMR data
Metabolic profiling of MH-Bm and MH-Ox media. The differences in the susceptibility of TB08-2-14 to SXT on MHF-Ox and MHF-Bm media suggest differences in the composition of MH media from these different suppliers. To investigate this, we first examined the composition of MH base medium from each supplier using nuclear magnetic resonance (NMR) spectroscopy, revealing differences in the composition of MH base medium, with the MH-Ox medium containing higher levels of adenine, uracil and TRIS, while the MH-Bm medium contained higher levels of uridine, glucose and an unknown guanine moiety containing compound. However, we were unable to identify any major differences in folate pathway compounds. To further investigate the composition of MH-Ox and MH-Bm, untargeted metabolomics analysis was performed using LC-MS. As with NMR, LC-MS data showed differences in the medium following positive and negative ionisation, but did not detect differences in folate pathway intermediates. Untargeted LC-MS profiling followed by multivariate statistical analysis (principal component analysis (PCA) and orthogonal projections to latent structures discriminant analysis (OPLS-DA) and database peak annotation revealed significant differences in a number of analytes, including higher levels of guanosine, uridine, methionine and peptides Pro-Ile-Ile and Pro-Val-Ile in the MH-Bm medium, with the MH-Ox medium containing higher levels of adenine, uracil, pyrrolidonecarboxylic acid and citric acid. Further comparison of spectra against reference standards for folate pathway intermediates revealed no significant differences other than a slight increase in 5,10-methylene-THF in MH-Bm. Thus, while there were major differences in the gross composition of MH media from each supplier, we were unable to identify substantial differences in the concentration of folic acid or reduced folate derivatives that might explain the relative performance of each media for detecting ThfT-mediated SMX resistance. 1H NMR sample preparation: 1H NMR was performed on a 600 MHz Bruker Avance III HD spectrometer, equipped with a 5 mm BBI probe and fitted with a Bruker SampleCase set to 6 °C. Time domain data were Fourier transformed and processed manually using Bruker TopspinTM 3.6.2 or Bruker TopspinTM 4.1.3 to obtain phase and baseline corrected spectra. Standard preparation. Stock solutions were prepared at 1 mg/mL in DMSO, and further diluted to 10 ug/mL in methanol containing 0.1 % butylated hydroxytolunene (BHT) for LC-MS analysis. Sample preparation. Media samples were filtered and either directly injected for the detection of targeted tetrahydrofolic acid metabolites, or diluted 1 in 10 (v/v) with LC-MS grade water for untargeted profiling. Preparation was identical for both methods.
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NMR: The cell media were thawed at 6 °C for 2 h and 560 µL directly injected into the NMR tube with 40 µL of D2O added and mixed thoroughly. All samples were prepared in standard 5 mm outer diameter NMR tubes. NMR spectroscopic analyses were performed on a 600 MHz Bruker Avance III HD spectrometer, equipped with a 5 mm BBI probe and fitted with a Bruker SampleCase set to 6 °C. All spectra were acquired at 300 K and the correct temperature was ensured with a 99.8% Methanol-d4 sample prior to measurement. The standard one-dimensional (1D) experiments with solvent suppression (pp: noesygppr1d) were acquired with 128 scans (+4 dummy scans), 64k data points, relaxation delay of 4.0 s, and a spectral width of 20 ppm resulting in a total experiment time of 14 min 53s. The spiking experiments were acquired with under the same conditions after addition of small quantities of the compound in question. Time domain data were Fourier transformed and processed manually using Bruker TopspinTM 3.6.2 or Bruker TopspinTM 4.1.3 to obtain phase and baseline corrected spectra. An exponential line broadening of 0.3 Hz was applied to the free induction decays (FID). LCMS: Stock solutions were prepared at 1 mg/mL in DMSO, and further diluted to 10 ug/mL in methanol containing 0.1 % butylated hydroxytolunene (BHT) for LC-MS analysis. Media samples were filtered and either directly injected for the detection of targeted tetrahydrofolic acid metabolites, or diluted 1 in 10 (v/v) with LC-MS grade water for untargeted profiling. Preparation was identical for both methods. Chromatographic separation was performed using a Waters Cortecs T3, 100mm x 2.1mm x 1.6 µm at 40 ºC. Mobile phase A was water + 0.1 % formic acid, and mobile phase B was acetonitrile + 0.1 % formic acid. The flow rate was 0.25 mL/min. Gradient elution was performed starting at 0 % B, held for 1.25 minutes, increasing to 10 % B at 6.00 minutes, 25 % B at 8.00 minutes and 75 % B at 10.50 minutes, followed by a wash step to 95 % B until 11.50 minutes. At 11.50 minutes, the flow was returned to initial conditions (0% B), allowing for 3.50 minutes of re-equilibration time. A 10 µL injection volume was performed. Mass spectrometry was performed on a Bruker Impact II QTOF-MS with electrospray ionisation operated in positive mode. The capillary voltage was 4500V, drying gas 10 L/min, gas temperature 220 ºC, nebuliser pressure 2.2 bar and the end plate offset 500 V. MS1 scan rate was set at 12 Hz. Auto MS/MS was enabled, with 4 precursors automatically selected and data collected at a scan rate of 25 Hz per scan cycle, resulting in a total scan cycle time (MS1 + auto MS/MS) of 0.2 seconds. An internal calibration was performed by injection of 5 mM sodium formate solution in water:isopropanol (50:50 v/v) at the beginning of every run. Data were collected with Compass HyStar 5.1 and O-TOF Control version 5.2, reviewed using Compass DataAnalysis 5.2 (Bruker Daltonics) and pre-processed in Metaboscape 2022 B.