RESUMEN

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.

Asunto(s)

Farmacorresistencia Fúngica , Antagonistas del Ácido Fólico , Metotrexato , Mutación , Pneumocystis carinii , Tetrahidrofolato Deshidrogenasa , Tetrahidrofolato Deshidrogenasa/genética , Tetrahidrofolato Deshidrogenasa/metabolismo , Tetrahidrofolato Deshidrogenasa/química , Pneumocystis carinii/genética , Pneumocystis carinii/enzimología , Pneumocystis carinii/efectos de los fármacos , Antagonistas del Ácido Fólico/farmacología , Farmacorresistencia Fúngica/genética , Metotrexato/farmacología , Regulación Alostérica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/efectos de los fármacos , Humanos , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/química , Dominio Catalítico/genética

RESUMEN

The lateral hypothalamus (LH) plays a critical role in sensory integration to organize behavior responses. However, how projection-defined LH neuronal outputs dynamically transmit sensorimotor signals to major downstream targets to organize behavior is unknown. Here, using multi-fiber photometry, we show that three major LH neuronal outputs projecting to the dorsal raphe nucleus (DRN), ventral tegmental area (VTA), and lateral habenula (LHb) exhibit significant coherent activity in mice engaging sensory-evoked or self-initiated motor responses. Increased activity at LH axon terminals precedes movement initiation during active coping responses and the activity of serotonin neurons and dopamine neurons. The optogenetic activation of LH axon terminals in either of the DRN, VTA, or LHb was sufficient to increase motor initiation but had different effects on passive avoidance and sucrose consumption. Our findings support the complementary role of three projection-defined LH neuronal outputs in the transmission of sensorimotor signals to major downstream regions at movement onset.