Dataset Information

Dual-Target Inhibitors of the Folate Pathway Inhibit Intrinsically Trimethoprim-Resistant DfrB Dihydrofolate Reductases.

ABSTRACT: Trimethoprim (TMP) is widely used to treat infections in humans and in livestock, accelerating the incidence of TMP resistance. The emergent and largely untracked type II dihydrofolate reductases (DfrBs) are intrinsically TMP-resistant plasmid-borne Dfrs that are structurally and evolutionarily unrelated to chromosomal Dfrs. We report kinetic characterization of the known DfrB family members. Their kinetic constants are conserved and all are poorly inhibited by TMP, consistent with TMP resistance. We investigate their inhibition with known and novel bisubstrate inhibitors of 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK). Importantly, all are inhibited by the HPPK inhibitors, making these molecules dual-target inhibitors of two folate pathway enzymes that are strictly microbial.

SUBMITTER: Toulouse JL

PROVIDER: S-EPMC7667824 | biostudies-literature | 2020 Nov

REPOSITORIES: biostudies-literature

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Dual-Target Inhibitors of the Folate Pathway Inhibit Intrinsically Trimethoprim-Resistant DfrB Dihydrofolate Reductases.

Toulouse Jacynthe L JL Shi Genbin G Lemay-St-Denis Claudèle C Ebert Maximilian C C J C MCCJC Deon Daniel D Gagnon Marc M Ruediger Edward E Saint-Jacques Kévin K Forge Delphine D Vanden Eynde Jean Jacques JJ Marinier Anne A Ji Xinhua X Pelletier Joelle N JN

ACS medicinal chemistry letters 20200928 11

Trimethoprim (TMP) is widely used to treat infections in humans and in livestock, accelerating the incidence of TMP resistance. The emergent and largely untracked type II dihydrofolate reductases (DfrBs) are intrinsically TMP-resistant plasmid-borne Dfrs that are structurally and evolutionarily unrelated to chromosomal Dfrs. We report kinetic characterization of the known DfrB family members. Their kinetic constants are conserved and all are poorly inhibited by TMP, consistent with TMP resistanc ...[more]

PMID: 33214838

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Project description:Enterococci are usually susceptible in vitro to trimethoprim; however, high-level resistance (HLR) (MICs, >1,024 microg/ml) has been reported. We studied Enterococcus faecalis DEL, for which the trimethoprim MIC was >1,024 microg/ml. No transfer of resistance was achieved by broth or filter matings. Two different genes that conferred trimethoprim resistance when they were cloned in Escherichia coli (MICs, 128 and >1,024 microg/ml) were studied. One gene that coded for a polypeptide of 165 amino acids (MIC, 128 microg/ml for E. coli) was identical to dfr homologs that we cloned from a trimethoprim-susceptible E. faecalis strain, and it is presumed to be the intrinsic E. faecalis dfr gene (which causes resistance in E. coli when cloned in multiple copies); this gene was designated dfrE. The nucleotide sequence 5' to this dfr gene showed similarity to thymidylate synthetase genes, suggesting that the dfr and thy genes from E. faecalis are located in tandem. The E. faecalis gene that conferred HLR to trimethoprim in E. coli, designated dfrF, codes for a predicted polypeptide of 165 amino acids with 38 to 64% similarity with other dihydrofolate reductases from gram-positive and gram-negative organisms. The nucleotide sequence 5' to dfrF did not show similarity to the thy sequences. A DNA probe for dfrF hybridized under high-stringency conditions only to colony lysates of enterococci for which the trimethoprim MIC was >1,024 microg/ml; there was no hybridization to plasmid DNA from the strain of origin. To confirm that this gene causes trimethoprim resistance in enterococci, we cloned it into the integrative vector pAT113 and electroporated it into RH110 (E. faecalis OG1RF::Tn916DeltaEm) (trimethoprim MIC, 0.5 microg/ml), which resulted in RH110 derivatives for which the trimethoprim MIC was >1, 024 microg/ml. These results indicate that dfrF is an acquired but probably chromosomally located gene which is responsible for in vitro HLR to trimethoprim in E. faecalis.

Project description:Streptococcus pneumoniae isolates resistant to several antimicrobial agent classes including trimethoprim-sulfamethoxazole have been reported with increasing frequency throughout the world. The MICs of trimethoprim, sulfamethoxazole, and trimethoprim-sulfamethoxazole (1:19) for 259 clinical isolates from South Africa were determined, and 166 of these 259 (64%) isolates were resistant to trimethoprim-sulfamethoxazole (MICs > or =20 mg/liter). Trimethoprim resistance was found to be more strongly correlated with trimethoprim-sulfamethoxazole resistance (correlation coefficient, 0.744) than was sulfamethoxazole resistance (correlation coefficient, 0.441). The dihydrofolate reductase genes from 11 trimethoprim-resistant (MICs, 64 to 512 microg/ml) clinical isolates of Streptococcus pneumoniae were amplified by PCR, and the nucleotide sequences were determined. Two main groups of mutations to the dihydrofolate reductase gene were found. Both groups shared six amino acid changes (Glu20-Asp, Pro70-Ser, Gln81-His, Asp92-Ala, Ile100-Leu, and Leu135-Phe). The first group included two extra changes (Lys60-Gln and Pro111-Ser), and the second group was characterized by six additional amino acid changes (Glu14-Asp, Ile74-Leu, Gln91-His, Glu94-Asp, Phe147-Ser, and Ala149-Thr). Chromosomal DNA from resistant isolates and cloned PCR products of the genes encoding resistant dihydrofolate reductases were capable of transforming a susceptible strain of S. pneumoniae to trimethoprim resistance. The inhibitor profiles of recombinant dihydrofolate reductase from resistant and susceptible isolates revealed that the dihydrofolate reductase from trimethoprim-resistant isolates was 50-fold more resistant (50% inhibitory doses [ID50s], 3.9 to 7.3 microM) than that from susceptible strains (ID50s, 0.15 microM). Site-directed mutagenesis experiments revealed that one mutation, Ile100-Leu, resulted in a 50-fold increase in the ID50 of trimethoprim. The resistant dihydrofolate reductases were characterized by highly conserved redundant changes in the nucleotide sequence, suggesting that the genes encoding resistant dihydrofolate reductases may have evolved as a result of inter- or intraspecies recombination by transformation.

Dataset Information

Dual-Target Inhibitors of the Folate Pathway Inhibit Intrinsically Trimethoprim-Resistant DfrB Dihydrofolate Reductases.

Publications

Dual-Target Inhibitors of the Folate Pathway Inhibit Intrinsically Trimethoprim-Resistant DfrB Dihydrofolate Reductases.

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