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

Abstract

dPneumocystis jirovecii is 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 of P. jirovecii DHFR 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, with K i values for trimethoprim 4-fold to 100-fold higher than those for the wild-type P. jirovecii DHFR. 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 increased K m values for dihydrofolic acid (DHFA). The catalytic rate constant (k cat ) was unchanged for most variant forms of P. jirovecii DHFR but was significantly lowered in F36C protein; one naturally occurring variant with two amino acid substitutions (S106P and E127G) showed a doubling of k cat , as well as a K m for 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 from P. jirovecii produce variant forms of DHFR that are resistant to trimethoprim and may contribute to clinically observed failures of standard therapy or prophylaxis.

Review of NAD(P)H-dependent oxidoreductases: Properties, engineering and application

“…In a recent study, six variants with a single amino acid substitution of Pneumocystis jirovecii DHFR were found to confer resistance against trimethoprim. An experimental drug also targeting DHFR but with a higher degree of conformational flexibility than trimethoprim (known as OAAG324) was tested against these variants, and found to be effective with one of them [116].…”

A Quantitative Model to Estimate Drug Resistance in Pathogens

“…To choose a quantitative model that demonstrates the strongest correlation to experimental data, we used kinetic data for the inhibition of PjDHFR variants with TMP [17]. This is the largest dataset available for drug targets and their variants in P. jirovecii .…”

“…Later, as the number of PCP patients unresponsive to TMP-SMX increased and the corresponding strains of the pathogen were sequenced, it became possible to draw statistically significant associations and estimate possible risks of resistance upon prior exposure to the drug [5–11]. Finally, in vitro enzymatic assays and PjDHPS/PjDHFR heterologous systems based on the respective knockouts in Saccharomyces cerevisiae enabled the measurement of the kinetic parameters of these enzymes with the wild type sequence and identified mutations [12–17]. …”

“…Moreover, our model can compute the effect of multi-position variants by considering them simultaneously. The new model was trained on the kinetics data of the PjDHFR inhibition by TMP and was further evaluated using experimental data for a different inhibitor targeting PjDHFR (OAAG324 [17]) as well as inhibition data for PjDHPS, Staphylococcus aureus DHFR, to estimate generalization of the model to different drugs, drug targets, and organisms.…”

Successful treatment of severe Pneumocystis pneumonia in an immunosuppressed patient using caspofungin combined with clindamycin: a case report and literature review

“…TMP-SMZ, an antibiotic used to treat a variety of infections, is the first-line drug for PCP prophylaxis and treatment, but treatment failure may occur because of dihydropteroate synthase and dihydrofolate reductase mutations during the course of the treatment [19, 20]. Adverse effects of TMP-SMZ are also common, such as fever, rash, nausea, vomiting, transaminase elevation, and more serious toxicities, including neutropenia, thrombocytopenia, Stevens-Johnson syndrome, and toxic epidermal necrolysis [21].…”