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

The dihydrofolate reductase (DHFR) is a drug target in many diseases, including infectious ones. Pneumocystis jirovecii is a fungal pathogen that causes pneumocystis pneumonia, a disease that mainly affects immunocompromised patients. This fungus has been historically hard to study because of our inability to grow it in vitro. One of the main drug targets in P. jiroveci is the DHFR (PjDHFR). Here, by using functional complementation of the baker's yeast DHFR with PjDHFR, we show that PjDHFR can be inhibited by methotrexate in a dose-dependent manner. By using deep mutational scanning of PjDHFR, we identify mutations that confer resistance to the antifolate methotrexate. 31 sites across the protein have at least one mutation that leads to resistance for a total of 603 high-confidence resistance mutations. Most sites where resistant mutations are found are inside the ligand or cofactor binding pockets, 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 lead to resistance. Surprisingly, one of these permissive sites is without direct contact to either ligand nor cofactor, suggesting that it acts through an allosteric mechanism. A structurally equivalent position had previously been identified as being putatively allosteric in bacterial DHFR. This evidence points towards a more important role of this position in DHFR function and 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 the effects of mutations in the main drug target of an uncultivable fungal pathogen.