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Antimicrobial Agents and Chemotherapy, April 2001, p. 1104-1108, Vol. 45, No. 4
0066-4804/01/$04.00+0   DOI: 10.1128/AAC.45.4.1104-1108.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

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

Jeffrey P. Maskell,* Armine M. Sefton, and 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

Received 8 August 2000/Returned for modification 12 November 2000/Accepted 12 January 2001

Trimethoprim resistance in Streptococcus pneumoniae can 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, Km values 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.

* Corresponding author. Mailing address: Department of Medical Microbiology, St. Bartholomew's and the Royal London School of Medicine and Dentistry, Turner St., Whitechapel, London E1 2AD, United Kingdom. Phone: (44) 020 7377 7257. Fax: (44) 020 7375 0518. E-mail: J.Maskell{at}mds.qmw.ac.uk.

Antimicrobial Agents and Chemotherapy, April 2001, p. 1104-1108, Vol. 45, No. 4
0066-4804/01/$04.00+0   DOI: 10.1128/AAC.45.4.1104-1108.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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