Multiple mutations modulate the function of dihydrofolate reductase in trimethoprim-resistant Streptococcus pneumoniae

Trimethoprim resistance in Streptococcus pneumoniae can be conferred by a s ingle amino acid substitution (I100-L) in dihydrofolate reductase (DHFR), b ut resistant clinical isolates usually carry multiple DHFR mutations. DHFR genes from five trimethoprim-resistant isolates from the United Kingdom wer e compared to susceptible isolates and used to transform a susceptible cont rol strain (CP1015). All trimethoprim-resistant isolates and transformants contained the I100-L mutation, The properties of DHFRs from transformants w ith different combinations of mutations were compared. In a transformant wi th 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 trime thoprim inhibition (50% inhibitory concentration [IC50], 4.2 muM) than was the DHFR from strain CP1015 (IC50, 0.09 muM) However, K-m values indicated a Lower affinity for the enzyme's natural substrates (K-m for dihydrofolate [DHF], 3.1 muM for CP1015 and 27.5 muM for R12/T2) and a twofold decrease in the specificity constant, In transformants with additional mutations in the C-terminal portion of the enzyme, K-m, values for DHF were reduced (9.2 to 15.2 muM), indicating compensation for the lower affinity generated by I100-L, Additional mutations in the N-terminal portion of the enzyme were a ssociated with up to threefold-increased resistance to trimethoprim (IC50, of up to 13.7 muM). It is postulated that carriage of the mutation M53-I-wh ich, like I100-L, corresponds to a trimethoprim binding site in the Escheri chia coli DHFR-is responsible for this increase. This study demonstrates th at although the I100-L mutation alone may give rise to trimethoprim resista nce, additional mutations serve to enhance resistance and modulate the effe cts of existing mutations on the affinity of DHFR for its natural substrate s.