Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:1.5.1.3 (dihydrofolate reductase)
 5,819 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We characterized a highly purified preparation of the chromosomally encoded dihydrofolate reductase (DHFR) from a trimethoprim-susceptible (Tmp8; strain MAP) and two trimethoprim-resistant (TmpR) strains (MAP/47 and MAP/42) of Haemophilus influenzae. The enzymes were purified between 650- and 3000-fold by gel-filtration and dye-ligand chromatography. The apparent molecular mass of the three proteins was 18400 Da by PAGE under denaturing and nondenaturing conditions. Total enzyme activity was greater in all fractions from the TmpR strains compared with the Tmp8 isolate. The three enzymes had a similar Km for dihydrofolate (7, 9 and 5 microM) and NADPH (2, 5 and 6 microM). However, the Tmp IC50 (the concentration necessary for 50% inhibition of DHFR activity) for the Tmp8 strain MAP was 0.001 microM, whereas DHFR from the TmpR strains MAP/47 and MAP/42 had values of 0.1 microM and 0.3 microM respectively. The methotrexate IC50 of the MAP/42 DHFR was 0.06 microM in comparison with the enzyme from MAP (0.008 microM) and MAP/47 (0.007 microM). Isoelectric focusing indicated that the DHFR from MAP/42 had a different isoelectric point (pI 7.6) compared with the enzymes from MAP and MAP/47 (pI 7.3). Peptide mapping after digestion with trypsin revealed one major peptide fragment (7.9 kDa) in the DHFR of MAP and MAP/47 and three major tryptic fragments (7.9, 9.6 and 12.5 kDa) in DHFR from MAP/42. We conclude that trimethoprim resistance in H. influenzae results from overproduction of structurally altered DHFR(s).
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PMID:Trimethoprim resistance in Haemophilus influenzae is due to altered dihydrofolate reductase(s). 201 95

We studied 10 trimethoprim-resistant (Tmpr) Haemophilus influenzae isolates for which agar dilution MICs were 10 to greater than 200 micrograms/ml. Trimethoprim resistance was transferred from two Tmpr H. influenzae isolates to a Tmps strain by conjugation or transformation. Wild-type Tmpr strains and Tmpr transcipients did not contain detectable plasmid DNA. The trimethoprim resistance gene was cloned into a cosmid vector, and recombinant plasmids were transduced into Escherichia coli. A 0.50-kilobase intragenic probe derived from a 12.9-kilobase fragment which encoded trimethoprim resistance hybridized with whole-cell DNA from Tmps and Tmpr strains. Southern blot analysis of restricted DNA from isogenic Tmps and Tmpr H. influenzae indicated that acquisition of trimethoprim resistance involved a rearrangement or change in nucleotide sequence. Hybridization was not seen with DNA derived from Tmpr E. coli containing dihydrofolate reductase I, II, and III genes or with Tmpr Neisseria meningitidis, Neisseria gonorrhoeae, and Pseudomonas cepacia. Southern hybridization with 12 multiply resistant encapsulated H. influenzae strains confirmed that the trimethoprim resistance gene was chromosomally mediated. Dihydrofolate reductase activity was significantly greater in cell sonicate supernatants of Tmpr strains in comparison with isogenic Tmps recipients. Differences were not found in the trimethoprim inhibition profile of dihydrofolate reductase activity in Tmps and Tmpr strains. We conclude that the mechanism of trimethoprim resistance in H. influenzae is overproduction of chromosomally located dihydrofolate reductase.
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PMID:Molecular cloning and mechanism of trimethoprim resistance in Haemophilus influenzae. 283 38

We previously demonstrated that trimethoprim (Tmp) resistance in Haemophilus influenzae is mediated by chromosomally encoded dihydrofolate reductase (DHFR) with a modified primary structure and distinct kinetic properties. To gain insight into the relationship of the DHFR structure and the level of Tmp resistance that it confers on the host bacterium, we cloned and characterized the folH genes of one Tmp-susceptible and two Tmp-resistant H. influenzae strains. Differences were observed between Tmp-susceptible and Tmp-resistant isolates both in the promoter region and in the coding sequences. The effect of differences between H. influenzae folH genes on Tmp susceptibility was investigated in Escherichia coli. Various folH gene hybrids were constructed, and their influence on Tmp susceptibility was determined. Resistance in E. coli mediated by folH from H. influenzae strain R1047 was associated with alterations in the promoter and the central part of folH. In contrast, the E. coli Tmp resistance phenotype associated with the folH gene of H. influenzae R1042 was characterized by alterations in one or more of three amino acid residues at the C-terminal part of the protein. These data indicate that Tmp resistance is not only related to alterations in the promoter region of the folH gene and the Tmp binding domains at the N-terminal and central part of DHFR. Alterations in the C-terminal part may also cause Tmp resistance, probably as a result of a change in secondary structure and the subsequent loss of Tmp binding affinity.
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PMID:Genetic characterization of trimethoprim resistance in Haemophilus influenzae. 887 94

In the last few decades, co-trimoxazole (SXT), an antibacterial combination of trimethoprim and sulfamethoxazole, has been used for treatment of upper respiratory tract infection due to Haemophilus influenzae. The usage of this antibiotic has become less important due to emergence of SXT-resistant strains worldwide. Most reports associate SXT resistance to the presence of variants of dihydrofolate reductase (DHFR) dfrA genes which are responsible for trimethoprim resistance; while the sulfamethoxazole (SMX) resistance are due to sulfonamide (SUL) genes sul1 and sul2 and/or mutation in the chromosomal (folP) gene encoding dihydropteroate synthetase (DHPS). This study aims to detect and analyse the genes that are involved in SXT resistance in H. influenzae strains that were isolated in Malaysia. Primers targeting for variants of dfrA, fol and sul genes were used to amplify the genes in nine SXT-resistant strains. The products of amplification were sequenced and multiple alignments of the assembled sequences of the local strains were compared to the sequences of other H. influenzae strains in the Genbank. Of the five variants of the dhfA genes, dfrA1 was detected in three out of the nine strains. In contrast to intermediate strains, at least one variant of folP genes was detected in the resistant strains. Multiple nucleotide alignment of this gene revealed that strain H152 was genetically different from the others due to a 15-bp nucleotide insert in folP gene. The sequence of the insert was similar to the insert in folP of H. influenzae strain A12, a strain isolated in United Kingdom. None of the strains had sul1 gene but sul2 gene was detected in four strains. Preliminary study on the limited number of samples shows that the TMP resistance was attributed to mainly to dfrA1 and the SMX was due to folP genes. Presence of sul2 in addition to folP in seven strains apparently had increased their level of resistance. A strain that lacked sul1 or sul2 gene, its resistance to sulfonamide was attributed to a 15-bp DNA insert in the folP gene.
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PMID:Molecular insights of co-trimoxazole resistance genes in Haemophilus influenzae isolated in Malaysia. 2452 26