Methylfolate Trap Promotes Bacterial Thymineless Death by Sulfa Drugs
Fig 5
Metabolic dynamics of the methylfolate trap in Salmonella typhimurium SULFA resistance.
(A) Wide-spectrum SULFA susceptibility of S. typhimurium metH(+) and metH(-) analyzed by 10X serial dilution. Cultures were diluted starting with OD1 and 5 μl cell suspensions were spotted onto LB agar in the absence (control) or presence of different SULFAs, used at the indicated concentrations. These SULFA drugs are classified into all four subgroups, in left-right order: short-acting (blue), intermediate-acting (yellow), long-acting (green), and ultra-long-acting (pink), respectively. Growth was recorded after 48 h at 37°C. (B) Viability of S. typhimurium metH(+) (red) and metH(-) (blue) on LB agar 24 h post-SMZ addition (125 μg/ml). Colony forming units (c.f.u.) were determined and normalized to c.f.u. values of the inoculation input (0 h). The y-axis represents c.f.u. fold-change on a log10 scale of SMZ-treated (+SMZ, hatched bars) and control non-treated samples (-SMZ, empty bars). Error bars represent standard deviations from biological triplicates. **** p<0.0001; ns, no significant difference. (C) SULFA susceptibility of S. typhimurium strains in liquid LB medium. Cultures of metH(+) (red) and metH(-) (blue) growing at OD1 was added with 2.5 mg/ml SMZ (arrow). Growth was monitored by measuring OD600. (D) Dynamics of the folate pool in S. typhimurium metH(+) (red) and metH(-) (blue) strains. At selected time points following SULFA treatment, cells were collected and folate extracted and analyzed by LC-MS/MS. Bars represent the combined levels of all 5-CH3-H4PteGlun species (top), all non-methylated folate species (middle), and total folate (bottom) following SMZ addition. s, significant difference between metH(-) and corresponding metH(+) samples; ns, no significant difference. (E) Dynamics of 41 metabolites in metH(+) (upper) and metH(-) (lower) strains. Metabolites are shown with their fold change over time (0–8 hours post SMZ addition). At selected time points following SMZ treatment, cells were collected and metabolites extracted and analyzed by LC-MS/MS. Signal intensity was normalized to OD600nm at each time point. Relative levels are expressed as the log ratio of the normalized signal intensity of SMZ-treated cells at each time point to the normalized signal intensity of the no drug control sample at t = 0 (n = 3).
The data shown in all figures represents the mean of biological repeats (n ≥ 3) with standard deviations. In the experiments demonstrated in Fig 5C–5E, SMZ was added at 2.5 mg/ml when cultures reached OD1.