TRIMETHOPRIM RESISTANCE IN HAEMOPHILUS-INFLUENZAE IS DUE TO ALTERED DIHYDROFOLATE REDUCTASE(S)

被引:18
作者
DEGROOT, R
CHAFFIN, DO
KUEHN, M
SMITH, AL
机构
[1] UNIV WASHINGTON, DEPT PEDIAT, SEATTLE, WA 98105 USA
[2] UNIV WASHINGTON, CHILDRENS ORTHOPED HOSP & MED CTR, DIV INFECT DIS, SEATTLE, WA 98105 USA
关键词
D O I
10.1042/bj2740657
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
We characterized a highly purified preparation of the chromosomally encoded dihydrofolate reductase (DHFR) from a trimethoprim-susceptible (Tmp(s); strain MAP) and two trimethoprim-resistant (Tmp(R)) 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 non-denaturing conditions. Total enzyme activity was greater in all fractions from the Tmp(R) strains compared with the Tmp(s) isolate. The three enzymes had a similar K(m) for dihydrofolate (7, 9 and 5-mu-M) and NADPH (2, 5 and 6-mu-M). However, the Tmp IC50 (the concentration necessary for 50 % inhibition of DHFR activity) for the Tmp(s) strain MAP was 0.001-mu-M, whereas DHFR from the Tmp(R) strains MAP/47 and MAP/42 had values of 0.1-mu-M and 0.3-mu-M respectively. The methotrexate IC50 of the MAP/42 DHFR was 0.06-mu-M in comparison with the enzyme from MAP (0.008-mu-M) and MAP/47 (0.007-mu-M). 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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页码:657 / 662
页数:6
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