A domino effect in antifolate drug action in Escherichia coli

Abstract

Mass spectrometry technologies for measurement of cellular metabolism are opening new avenues to explore drug activity. Trimethoprim is an antibiotic that inhibits bacterial dihydrofolate reductase (DHFR). Kinetic flux profiling with (15)N-labeled ammonia in Escherichia coli reveals that trimethoprim leads to blockade not only of DHFR but also of another critical enzyme of folate metabolism: folylpoly-gamma-glutamate synthetase (FP-gamma-GS). Inhibition of FP-gamma-GS is not directly due to trimethoprim. Instead, it arises from accumulation of DHFR's substrate dihydrofolate, which we show is a potent FP-gamma-GS inhibitor. Thus, owing to the inherent connectivity of the metabolic network, falling DHFR activity leads to falling FP-gamma-GS activity in a domino-like cascade. This cascade results in complex folate dynamics, and its incorporation in a computational model of folate metabolism recapitulates the dynamics observed experimentally. These results highlight the potential for quantitative analysis of cellular metabolism to reveal mechanisms of drug action.

Figure 1

Changes in folate pools with the addition of trimethoprim (4 μg ml⁻¹). As expected, the DHFR inhibitor caused an overall increase in oxidized folates and a decrease in reduced folates. Surprisingly, reduced mono- and diglutamate species increased in concentration after an initial decrease. The x axis represents minutes after drug addition, and the y axis represents μM concentrations on a log scale. The error bars show +1 s.e.m. (n = 3 independent experiments).

Figure 2

Probing folate fluxes in the presence of trimethoprim. Trimethoprim treatment decreases flux through folylpoly-γ-glutamate synthetase (FP-γ-GS) but does not affect flux through folylpoly-α-glutamate synthetase (FP-α-GS). Control E. coli cells were grown in ¹⁴N medium and then switched to ¹⁵N medium. Drug-treated cells were grown in ¹⁴N medium, treated with 4 μg ml⁻¹ trimethoprim for 15 min, and then switched to ¹⁵N medium also containing 4 μg ml⁻¹ trimethoprim. The x axis represents minutes after N-switch. In the top row, the y axis represents the percent of the indicated folate species containing greater than or equal to one ¹⁵N atom (any-labeled). In the bottom row, the y axis represents the percent containing greater than or equal to one ¹⁵N atom in the pteroate portion of the molecule (core structure-labeled). Error bars show ±1 s.e.m. (n = 3 independent experiments). Curves represent a fit of the data to equations presented previously as described in the Methods.

Figure 3

DHF inhibits FP-γ-GS activity, as determined by in vitro FP-γ-GS assays. (a) DHF (H₂PteGlu₁) inhibits FP-γ-GS activity, but trimethoprim (Tm), 5-methyl-H₄PteGlu₁ (MTHF) and folate (PteGlu₁) do not. H₄PteGlu₂ production was measured as peak intensity from LC-MS/MS analysis. (b) Dosedependent inhibition of FP-γ-GS activity by DHF. H₄PteGlu₁ consumption was measured as peak intensity from LC-MS/MS analysis. The line indicates a fit to competitive Michaelis-Menten kinetics with K_i = 3.1 μM. Error bars show ±1 s.e.m. (n = 3 independent experiments).

Figure 4

Computational modeling of cellular folate dynamics. (a) Model schematic. The model simulates reactions corresponding to folate oxidation/reduction, glutamate tail extension (by FP-γ-GS and FP-α-GS as appropriate) and degradation (from the oxidized state). One-carbon transfer reactions are not explicitly considered, as the observed flux data (Supplementary Fig. 4) indicate that they are uniformly faster than the glutamate chain extension reactions of interest. All simulated reactions were assumed to be first order with respect to their folate substrates, except for the pivotal FP-γ-GS reactions, which were modeled as Michaelis-Menten forms with competitive inhibition by H₂PteGlu₁ and H₂PteGlu₂ (similar results were obtained assuming inhibition by H₂PteGlu₁ only). The script is supplied in the Supplementary Methods. (b) The computational model shows the same qualitative behaviors as the experimental data. The (C₁)-H₄PteGlu_n data represent the sum of all measured reduced folates containing n glutamates. Error bars show ±1 s.e.m. (n = 3 independent experiments).

Scheme 1

Folate synthesis, one-carbon substitution, polyglutamation and catabolism in E. coli. A series of reactions converts GTP and pABA to dihydropteroic acid (H₂Pte). Linkage of H₂Pte to glutamate forms dihydrofolate (H₂PteGlu₁, DHF). DHF and its polyglutamated forms (H₂PteGlu_n) are reduced by DHFR to form tetrahydrofolate species (H₄PteGlu_n), which can gain one-carbon units to form C₁-H₄PteGlu_n (5-methyl-H₄PteGlu_n, 5-formyl-H₄PteGlu_n, 5-formimino-H₄PteGlu_n, 10-formyl-H₄PteGlu_n, 5,10-methenyl-H₄PteGlu_n and 5,10-methylene- H₄PteGlu_n), the active one-carbon donors in specific biosynthetic reactions. 5,10-methylene-H₄PteGlu_n is the substrate of TS, which regenerates H₂PteGlu_n. H₂PteGlu_n can also be oxidized to folate species (PteGlu_n) or catabolized to pABGlu_n. Both H₄PteGlu_n and C₁-H₄PteGlu_n are substrates of folylpolyglutamate synthetase (FPGS) to form the corresponding (C₁)- H₄PteGlu_n+1 species. Second and third glutamate residues are connected via γ-linkages to previous glutamates, and the rest are connected via α-linkages.