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

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 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 associated 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-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.