Trimethoprim resistance in Haemophilus influenzae is due to altered dihydrofolate reductase(s).
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).
de Groot
R
R
Division of Infectious Disease, Children's Hospital and Medical Center, Seattle, WA.
Chaffin
D O
DO
Kuehn
M
M
Smith
A L
AL
D002850:Chromatography, Gel; D004351:Drug Resistance; D004591:Electrophoresis, Polyacrylamide Gel; D006193:Haemophilus influenzae / Q000187:drug effects* / Q000201:enzymology; D007525:Isoelectric Focusing; D007700:Kinetics; D009154:Mutation; D010449:Peptide Mapping; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics*; D014295:Trimethoprim / Q000494:pharmacology*
D014295:Trimethoprim; D013762:Tetrahydrofolate Dehydrogenase