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

Abstract

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 [IC 50 ], 4.2 M) than was the DHFR from strain CP1015 (IC 50 , 0.09 M). However, K m values indicated a lower affinity for the enzyme's natural substrates (K m for dihydrofolate [DHF], 3.1 M for CP1015 and 27.5 M 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 M), 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 (IC 50 of up to 13.7 M). It is postulated that carriage of the mutation M53-Iwhich, 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.

Biological and Epidemiological Features of Antibiotic-Resistant Streptococcus pneumoniae in Pre- and Post-Conjugate Vaccine Eras: a United States Perspective

“…Trimethoprim resistance in pneumococci results from a single amino acid substitution (Ile100¡Leu) in the dihydrofolate reductase (DHFR) protein (225) encoded by folA and is often associated with mosaic alleles. Additional mutations have been reported, which appear to modulate these alterations, affecting the affinity of DHFR for its natural substrates and thereby enhancing resistance (226). Resistance to sulfonamides is most often associated with localized 1-or 2-codon insertion mutations within the folP gene encoding dihydropteroate synthase (DHPS).…”

Antibiotic Resistance Markers in Burkholderia pseudomallei Strain Bp1651 Identified by Genome Sequence Analysis

“…There were two substitutions in DHFR (FolA) in Bp1651 TR70_1420, I99L and A145T, in comparison to three other strains. The I99L substitution at the equivalent position occurs in trimethoprimresistant Streptococcus pneumoniae (I100L) and is sufficient to confer trimethoprim resistance in that species (56)(57)(58). This substitution is localized to the predicted active site of the DHFR enzymes (59) and may therefore be responsible for trimethoprim resistance in B. pseudomallei.…”

Mutations of Pneumocystis jirovecii Dihydrofolate Reductase Associated with Failure of Prophylaxis

“…Alignment of amino acid sequence of P. jirovecii DHFR with those from distantly related organisms. Positions of amino acid changes that confer resistance to PM or TMP in P. falciparum (15), S. pneumoniae (10), and S. aureus (4) are shown in boldface. Amino acid changes we found in P. jirovecii are also shown in boldface.…”

“…Alteration of DHFR enzyme is a common resistance mechanism in clinically important microbial pathogens, such as Plasmodium falciparum (15) and Streptococcus pneumoniae (10). Two studies have investigated the possibility of mutations in P. jirovecii DHFR gene.…”