Mechanism of the antimicrobial drug trimethoprim revisited

Abstract

We tested the hypothesis that the mechanism of action of the antifolate drug trimethoprim is through accumulation of bacterial dihydrofolate resulting in depletion of tetrahydrofolate coenzymes required for purine and pyrimidine biosynthesis. The folate pool of a strain of Escherichia coli (NCIMB 8879) was prelabeled with the folate biosynthetic precursor [(3)H]-p-aminobenzoic acid before treatment with trimethoprim. Folates in untreated E. coli were present as tetrahydrofolate coenzymes. In trimethoprim-treated cells, however, a rapid transient accumulation of dihydrofolate occurred, followed by complete conversion of all forms of folate to cleaved catabolites (pteridines and para-aminobenzoylglutamate) and the stable nonreduced form of the vitamin, folic acid. Both para-aminobenzoylglutamate and folic acid were present in the cell in the form of polyglutamates. Removal of trimethoprim resulted in the reconversion of the accumulated folic acid to tetrahydrofolate cofactors for subsequent participation in the one-carbon cycle. Whereas irreversible catabolism is probably bactericidal, conversion to folic acid may constitute a bacteriostatic mechanism since, as we show, folic acid can be used by the bacteria and proliferation is resumed once trimethoprim is removed. Thus, the clinical effectiveness of this important drug may depend on the extent to which the processes of either catabolism or folic acid production occur in different bacteria or during different therapeutic regimes.

Isotope ratio-based profiling of microbial folates

“…In addition, the method was used to characterize the response of E. coli folate pools to the dihydrofolate reductase (DHFR) inhibitor trimethoprim. In addition to recapitulating literature findings regarding accumulation of oxidized and depletion of reduced folates in trimethoprim-treated E. coli [12], we demonstrate that trimethoprim treatment leads to profound decreases in folate polyglutamate chain length. This observation, which would not have been feasible using methods focused only on monoglutamated folates, highlights the value of LC-MS/MS methods that examine the full spectrum of cellular folate species.…”

“…All but one of the 27 folate-related species that we quantified successfully changed substantially (Ͼ2-fold and P Ͻ 0.05) in concentration in response to the drug. Certain of these responses were expected based on literature knowledge of the drug's mechanism of action and effect on cellular folate pools [12]: in general, tetrahydrofolate species decreased and dihydrofolate (or yet more oxidized) species increased (in Table 6, this is reflected as values Ͼ1 predominating in the top half of the table and Ͻ1 predominating in the lower half of the table). These results confirm the ability of our LC-MS/MS method to recapitulate a well characterized pharmacological response.…”

“…The antimicrobial drug trimethoprim is an inhibitor of DHFR [12,25]. Combined with sulfonamide, it is used clinically to treat diseases caused by enteric bacteria such as urinary tract infections.…”

Escherichia coli abg Genes Enable Uptake and Cleavage of the Folate Catabolite p -Aminobenzoyl-Glutamate

“…A report from Quinlivan et al has provided some insights into E. coli folate metabolism under stress (20). Using highperformance liquid chromatography analytic techniques, these authors investigated the mechanism of the antibacterial drug trimethoprim.…”

Examination of intrinsic sulfonamide resistance in Bacillus anthracis: A novel assay for dihydropteroate synthase

“…The tetrahydrofolate produced by this pathway is required for one carbon transfer reactions in the biosynthesis of biomolecules including nucleotides and amino acids [1][2]. Humans lack an equivalent to DHPS, making it an attractive drug target.…”