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Trimethoprim resistance in Haemophilus influenzae is due to altered dihydrofolate reductase(s)
R de Groot1, D O Chaffin, M Kuehn
1Division of Infectious Disease, Children's Hospital and Medical Center, Seattle, WA.
Abstract:
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).
Insights
Trimethoprim resistance in Haemophilus influenzae is linked to altered dihydrofolate reductase (DHFR) enzymes. These resistant strains overproduce structurally different DHFR, impacting drug efficacy.
Area of Science:
- Microbiology
- Biochemistry
- Molecular Biology
Background:
- Dihydrofolate reductase (DHFR) is a crucial enzyme in folate metabolism.
- Trimethoprim is an antibiotic that targets DHFR.
- Antibiotic resistance is a growing public health concern.
Purpose of the Study:
- To characterize the chromosomally encoded dihydrofolate reductase (DHFR) from trimethoprim-susceptible and resistant strains of Haemophilus influenzae.
- To investigate the molecular basis of trimethoprim resistance in H. influenzae.
Main Methods:
- Enzyme purification using gel-filtration and dye-ligand chromatography.
- Enzyme activity assays, including Km determination and IC50 measurements for trimethoprim and methotrexate.
- Isoelectric focusing and peptide mapping to analyze enzyme structure.
Main Results:
- Purified DHFR from trimethoprim-resistant strains showed higher total enzyme activity.
- Resistant DHFR enzymes exhibited significantly higher IC50 values for trimethoprim compared to susceptible strains.
- Structural differences were observed in DHFR from resistant strains, including altered isoelectric points and tryptic peptide profiles.
Conclusions:
- Trimethoprim resistance in H. influenzae is associated with overproduction of structurally altered DHFR.
- These structural alterations likely contribute to reduced drug binding and confer resistance.
- Understanding these mechanisms is vital for developing strategies to combat antibiotic resistance.