Trimethoprim Resistance of Dihydrofolate Reductase Variants from Clinical Isolates of Pneumocystis jirovecii

Abstract

ABSTRACTPneumocystis jiroveciiis an opportunistic pathogen that causes serious pneumonia in immunosuppressed patients. Standard therapy and prophylaxis include trimethoprim (TMP)-sulfamethoxazole; trimethoprim in this combination targets dihydrofolate reductase (DHFR). Fourteen clinically observed variants ofP. jiroveciiDHFR were produced recombinantly to allow exploration of the causes of clinically observed failure of therapy and prophylaxis that includes trimethoprim. Six DHFR variants (S31F, F36C, L65P, A67V, V79I, and I158V) showed resistance to inhibition by trimethoprim, withKivalues for trimethoprim 4-fold to 100-fold higher than those for the wild-typeP. jiroveciiDHFR. An experimental antifolate with more conformational flexibility than trimethoprim showed strong activity against one trimethoprim-resistant variant. The two variants that were most resistant to trimethoprim (F36C and L65P) also had increasedKmvalues for dihydrofolic acid (DHFA). The catalytic rate constant (kcat) was unchanged for most variant forms ofP. jiroveciiDHFR but was significantly lowered in F36C protein; one naturally occurring variant with two amino acid substitutions (S106P and E127G) showed a doubling ofkcat, as well as aKmfor NADPH half that of the wild type. The strongest resistance to trimethoprim occurred with amino acid changes in the binding pocket for DHFA or trimethoprim, and the strongest effect on binding of NADPH was linked to a mutation involved in binding the phosphate group of the cofactor. This study marks the first confirmation that naturally occurring mutations in the gene for DHFR fromP. jiroveciiproduce variant forms of DHFR that are resistant to trimethoprim and may contribute to clinically observed failures of standard therapy or prophylaxis.