Stepwise Acquisition of Pyrimethamine Resistance in the Malaria Parasite

The spread of high-level pyrimethamine resistance in Africa threatens to curtail the therapeutic lifetime of antifolate antimalarials. We studied the possible evolutionary pathways in the evolution of pyrimethamine resistance using an approach in which all possible mutational intermediates were created by site-directed mutagenesis and assayed for their level of drug resistance. The coding sequence for dihydrofolate reductase (DHFR) from the malaria parasite Plasmodium falciparum was mutagenized, and tests were carried out in Escherichia coli under conditions in which the endogenous bacterial enzyme was selectively inhibited. We studied 4 key amino acid replacements implicated in pyrimethamine resistance: N51I, C59R, S108N, and I164L. Using empirical estimates of the mutational spectrum in P. falciparum and probabilities of fixation based on the relative levels of resistance, we found that the predicted favored pathways of drug resistance are consistent with those reported in previous kinetic studies, as well as DHFR polymorphisms observed in natural populations. We found that 3 pathways account for nearly 90% of the simulated realizations of the evolution of pyrimethamine resistance. The most frequent pathway (S108N and then C59R, N51I, and I164L) accounts for more than half of the simulated realizations. Our results also suggest an explanation for why I164L is detected in Southeast Asia and South America, but not at significant frequencies in Africa.Organismic and Evolutionary Biolog

Predictability of evolutionary trajectories in fitness landscapes

Experimental studies on enzyme evolution show that only a small fraction of all possible mutation trajectories are accessible to evolution. However, these experiments deal with individual enzymes and explore a tiny part of the fitness landscape. We report an exhaustive analysis of fitness landscapes constructed with an off-lattice model of protein folding where fitness is equated with robustness to misfolding. This model mimics the essential features of the interactions between amino acids, is consistent with the key paradigms of protein folding and reproduces the universal distribution of evolutionary rates among orthologous proteins. We introduce mean path divergence as a quantitative measure of the degree to which the starting and ending points determine the path of evolution in fitness landscapes. Global measures of landscape roughness are good predictors of path divergence in all studied landscapes: the mean path divergence is greater in smooth landscapes than in rough ones. The model-derived and experimental landscapes are significantly smoother than random landscapes and resemble additive landscapes perturbed with moderate amounts of noise; thus, these landscapes are substantially robust to mutation. The model landscapes show a deficit of suboptimal peaks even compared with noisy additive landscapes with similar overall roughness. We suggest that smoothness and the substantial deficit of peaks in the fitness landscapes of protein evolution are fundamental consequences of the physics of protein folding.Comment: 14 pages, 7 figure

Reciprocal Sign Epistasis between Frequently Experimentally Evolved Adaptive Mutations Causes a Rugged Fitness Landscape

The fitness landscape captures the relationship between genotype and evolutionary fitness and is a pervasive metaphor used to describe the possible evolutionary trajectories of adaptation. However, little is known about the actual shape of fitness landscapes, including whether valleys of low fitness create local fitness optima, acting as barriers to adaptive change. Here we provide evidence of a rugged molecular fitness landscape arising during an evolution experiment in an asexual population of Saccharomyces cerevisiae. We identify the mutations that arose during the evolution using whole-genome sequencing and use competitive fitness assays to describe the mutations individually responsible for adaptation. In addition, we find that a fitness valley between two adaptive mutations in the genes MTH1 and HXT6/HXT7 is caused by reciprocal sign epistasis, where the fitness cost of the double mutant prohibits the two mutations from being selected in the same genetic background. The constraint enforced by reciprocal sign epistasis causes the mutations to remain mutually exclusive during the experiment, even though adaptive mutations in these two genes occur several times in independent lineages during the experiment. Our results show that epistasis plays a key role during adaptation and that inter-genic interactions can act as barriers between adaptive solutions. These results also provide a new interpretation on the classic Dobzhansky-Muller model of reproductive isolation and display some surprising parallels with mutations in genes often associated with tumors

Accessible Mutational Trajectories for the Evolution of Pyrimethamine Resistance in the Malaria Parasite Plasmodium Vivax

Antifolate antimalarials, such as pyrimethamine, have experienced a dramatic reduction in therapeutic efficacy as resistance has evolved in multiple malaria species. We present evidence from one such species, Plasmodium vivax, which has experienced sustained selection for pyrimethamine resistance at the dihydrofolate reductase (DHFR) locus since the 1970s. Using a transgenic Saccharomyces cerevisiae model expressing the P. vivax DHFR enzyme, we assayed growth rate and resistance of all 16 combinations of four DHFR amino acid substitutions. These substitutions were selected based on their known association with drug resistance, both in natural isolates and in laboratory settings, in the related malaria species P. falciparum. We observed a strong correlation between the resistance phenotypes for these 16 P. vivax alleles and previously observed resistance data for P. falciparum, which was surprising since nucleotide diversity levels and common polymorphic variants of DHFR differ between the two species. Similar results were observed when we expressed the P. vivax alleles in a transgenic bacterial system. This suggests common constraints on enzyme evolution in the orthologous DHFR proteins. The interplay of negative trade-offs between the evolution of novel resistance and compromised endogenous function varies at different drug dosages, and so too do the major trajectories for DHFR evolution. In simulations, it is only at very high drug dosages that the most resistant quadruple mutant DHFR allele is favored by selection. This is in agreement with common polymorphic DHFR data in P. vivax, from which this quadruple mutant is missing. We propose that clinical dosages of pyrimethamine may have historically been too low to select for the most resistant allele, or that the fitness cost of the most resistant allele was untenable without a compensatory mutation elsewhere in the genome.Organismic and Evolutionary Biolog

Biophysical principles predict fitness landscapes of drug resistance

Fitness landscapes of drug resistance constitute powerful tools to elucidate mutational pathways of antibiotic escape. Here, we de- veloped a predictive biophysics-based fitness landscape of trimeth- oprim (TMP) resistance for Escherichia coli dihydrofolate reductase (DHFR). We investigated the activity, binding, folding stability, and intracellular abundance for a complete set of combinatorial DHFR mutants made out of three key resistance mutations and extended this analysis to DHFR originated from Chlamydia muridarum and Listeria grayi. We found that the acquisition of TMP resistance via decreased drug affinity is limited by a trade-off in catalytic efficiency. Protein stability is concurrently affected by the resistant mutants, which precludes a precise description of fitness from a single molec- ular trait. Application of the kinetic flux theory provided an accurate model to predict resistance phenotypes (IC50) quantitatively from a unique combination of the in vitro protein molecular properties. Fur- ther, we found that a controlled modulation of the GroEL/ES chap- eronins and Lon protease levels affects the intracellular steady-state concentration of DHFR in a mutation-specific manner, whereas IC50 is changed proportionally, as indeed predicted by the model. This un- veils a molecular rationale for the pleiotropic role of the protein quality control machinery on the evolution of antibiotic resistance, which, as we illustrate here, may drastically confound the evolution- ary outcome. These results provide a comprehensive quantitative genotype–phenotype map for the essential enzyme that serves as an important target of antibiotic and anticancer therapies.Organismic and Evolutionary Biolog