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Mechanisms of Resistance

Multiple Mutations Modulate the Function of Dihydrofolate Reductase in Trimethoprim-ResistantStreptococcus pneumoniae

Jeffrey P. Maskell, Armine M. Sefton, Lucinda M. C. Hall
Jeffrey P. Maskell
Department of Medical Microbiology, St. Bartholomew's and the Royal London School of Medicine and Dentistry, London E1 2AD, United Kingdom
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Armine M. Sefton
Department of Medical Microbiology, St. Bartholomew's and the Royal London School of Medicine and Dentistry, London E1 2AD, United Kingdom
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Lucinda M. C. Hall
Department of Medical Microbiology, St. Bartholomew's and the Royal London School of Medicine and Dentistry, London E1 2AD, United Kingdom
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DOI: 10.1128/AAC.45.4.1104-1108.2001
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ABSTRACT

Trimethoprim resistance in Streptococcus pneumoniaecan be conferred by a single amino acid substitution (I100-L) in dihydrofolate reductase (DHFR), but resistant clinical isolates usually carry multiple DHFR mutations. DHFR genes from five trimethoprim-resistant isolates from the United Kingdom were compared to susceptible isolates and used to transform a susceptible control strain (CP1015). All trimethoprim-resistant isolates and transformants contained the I100-L mutation. The properties of DHFRs from transformants with different combinations of mutations were compared. In a transformant with only the I100-L mutation (R12/T2) and a D92-A mutation also found in the DHFRs of susceptible isolates, the enzyme was much more resistant to trimethoprim inhibition (50% inhibitory concentration [IC50], 4.2 μM) than was the DHFR from strain CP1015 (IC50, 0.09 μM). However,Km values indicated a lower affinity for the enzyme's natural substrates (Km for dihydrofolate [DHF], 3.1 μM for CP1015 and 27.5 μM for R12/T2) and a twofold decrease in the specificity constant. In transformants with additional mutations in the C-terminal portion of the enzyme, Kmvalues for DHF were reduced (9.2 to 15.2 μM), indicating compensation for the lower affinity generated by I100-L. Additional mutations in the N-terminal portion of the enzyme were associated with up to threefold-increased resistance to trimethoprim (IC50 of up to 13.7 μM). It is postulated that carriage of the mutation M53-I—which, like I100-L, corresponds to a trimethoprim binding site in the Escherichia coli DHFR—is responsible for this increase. This study demonstrates that although the I100-L mutation alone may give rise to trimethoprim resistance, additional mutations serve to enhance resistance and modulate the effects of existing mutations on the affinity of DHFR for its natural substrates.

  • Copyright © 2001 American Society for Microbiology
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Multiple Mutations Modulate the Function of Dihydrofolate Reductase in Trimethoprim-ResistantStreptococcus pneumoniae
Jeffrey P. Maskell, Armine M. Sefton, Lucinda M. C. Hall
Antimicrobial Agents and Chemotherapy Apr 2001, 45 (4) 1104-1108; DOI: 10.1128/AAC.45.4.1104-1108.2001

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Multiple Mutations Modulate the Function of Dihydrofolate Reductase in Trimethoprim-ResistantStreptococcus pneumoniae
Jeffrey P. Maskell, Armine M. Sefton, Lucinda M. C. Hall
Antimicrobial Agents and Chemotherapy Apr 2001, 45 (4) 1104-1108; DOI: 10.1128/AAC.45.4.1104-1108.2001
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KEYWORDS

Anti-Infective Agents, Urinary
mutation
Streptococcus pneumoniae
Tetrahydrofolate Dehydrogenase
trimethoprim

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