Journal Article

Deep mutational scanning of Pneumocystis jirovecii dihydrofolate reductase reveals allosteric mechanism of resistance to an antifolate

Landry, Christian R., Després, Philippe C., Pageau, Alicia, Gagnon-Arsenault, Isabelle, Dubé, Alexandre K., Lagüe, Patrick, Dibyachintan, Soham, Rouleau, Francois D.

2024

Overview

Pneumocystis jirovecii is a fungal pathogen that causes pneumocystis pneumonia, a disease that mainly affects immunocompromised individuals. This fungus has historically been hard to study because of our inability to grow it in vitro . One of the main drug targets in P . jirovecii is its dihydrofolate reductase (PjDHFR). Here, by using functional complementation of the baker’s yeast ortholog, we show that PjDHFR can be inhibited by the antifolate methotrexate in a dose-dependent manner. Using deep mutational scanning of PjDHFR, we identify mutations conferring resistance to methotrexate. Thirty-one sites spanning the protein have at least one mutation that leads to resistance, for a total of 355 high-confidence resistance mutations. Most resistance-inducing mutations are found inside the active site, and many are structurally equivalent to mutations known to lead to resistance to different antifolates in other organisms. Some sites show specific resistance mutations, where only a single substitution confers resistance, whereas others are more permissive, as several substitutions at these sites confer resistance. Surprisingly, one of the permissive sites (F199) is without direct contact to either ligand or cofactor, suggesting that it acts through an allosteric mechanism. Modeling changes in binding energy between F199 mutants and drug shows that most mutations destabilize interactions between the protein and the drug. This evidence points towards a more important role of this position in resistance than previously estimated and highlights potential unknown allosteric mechanisms of resistance to antifolate in DHFRs. Our results offer unprecedented resources for the interpretation of mutation effects in the main drug target of an uncultivable fungal pathogen.

• Publisher: Public Library of Science,Public Library of Science (PLoS)
• Subject: Allosteric enzymes / Allosteric properties / Allosteric Regulation / Bacteria / Catalytic Domain - genetics / CRISPR / Dihydrofolate reductase / Disease prevention / DNA polymerase / Drug resistance / Drug resistance in microorganisms / Drug Resistance, Fungal - genetics / Enzymes / Folic Acid Antagonists - pharmacology / Fungal infections / Fungal Proteins - chemistry / Fungal Proteins - genetics / Fungal Proteins - metabolism / Gene mutations / Genetic aspects / Genomes / Humans / Methotrexate / Methotrexate - pharmacology / Microbial enzymes / Microbiological research / Mortality / Mutation / Pathogens / Physiological aspects / Plasmids / Pneumocystis carinii / Pneumocystis carinii - drug effects / Pneumocystis carinii - enzymology / Pneumocystis carinii - genetics / Pneumocystis jirovecii / Pneumonia / Proteins / Saccharomyces cerevisiae - drug effects / Saccharomyces cerevisiae - genetics / Scanning / Tetrahydrofolate Dehydrogenase - chemistry / Tetrahydrofolate Dehydrogenase - genetics / Tetrahydrofolate Dehydrogenase - metabolism / Therapeutic targets / Yeast