Compensatory Mutations Restore Fitness during the Evolution of Dihydrofolate Reductase

Abstract

Whether a trade-off exists between robustness and evolvability is an important issue for protein evolution. Although traditional viewpoints have assumed that existing functions must be compromised by the evolution of novel activities, recent research has suggested that existing phenotypes can be robust to the evolution of novel protein functions. Enzymes that are targets of antibiotics that are competitive inhibitors must evolve decreased drug affinity while maintaining their function and sustaining growth. Utilizing a transgenic Saccharomyces cerevisiae model expressing the dihydrofolate reductase (DHFR) enzyme from the malarial parasite Plasmodium falciparum, we examine the robustness of growth rate to drug-resistance mutations. We assay the growth rate and resistance of all 48 combinations of 6 DHFR point mutations associated with increased drug resistance in field isolates of the parasite. We observe no consistent relationship between growth rate and resistance phenotypes among the DHFR alleles. The three evolutionary pathways that dominate DHFR evolution show that mutations with increased resistance can compensate for initial declines in growth rate from previously acquired mutations. In other words, resistance mutations that occur later in evolutionary trajectories can compensate for the fitness consequences of earlier mutations. Our results suggest that high levels of resistance may be selected for without necessarily jeopardizing overall fitness.

Prospective Screening of Novel Antibacterial Inhibitors of Dihydrofolate Reductase for Mutational Resistance

“…Previous studies suggest that resistance mutations may affect bacterial fitness by affecting growth rates, survival rates, or catalysis of the enzyme drug target (3,25,37). In this study, we investigated the bacterial fitness of resistant strains containing the F98Y, F98I, and H30N genotypes.…”

Topological features of rugged fitness landscapes in sequence space

“…One way to test this prediction is to reconstruct the order of substitutions in which evolution proceeded and then to test whether accumulation of the substitutions in a different order is possible [45,[76][77][78][79][80][81][82]. A variation of this test was performed recently [82] for a nucleoprotein in the human H3N2 influenza virus.…”

Evolution of Plasmodium falciparum drug resistance: implications for the development and containment of artemisinin resistance

“…Dihydrofolate reductase (DHFR), which is encoded by the dhfr gene (28), reduces dihydrofolate to tetrahydrofolate, an essential cofactor in the synthesis of nucleic acids and methionine. Mutations at amino acid positions 50, 51, 59, 108, and 164 of DHFR are implicated in pyrimethamine resistance (31)(32)(33)(34)(35). A Ser-to-Asn substitution at position 108 (CNCNI, where letters indicate amino acid positions 50, 51, 59, 108, and 164, and the altered residues are underlined) is the first step for acquiring increased resistance.…”

“…A Ser-to-Asn substitution at position 108 (CNCNI, where letters indicate amino acid positions 50, 51, 59, 108, and 164, and the altered residues are underlined) is the first step for acquiring increased resistance. Overall, as the number of substitutions in DHFR increases, the level of pyrimethamine resistance becomes higher (31)(32)(33)(34)(35). At present, the quadruple mutant enzyme (CIRNL) shows the highest 50z inhibitory concentration (IC 50 ) for pyrimethamine.…”