Fitness costs, compensation and epistasis

Overview

We use CRISPR/Cas9 approaches to modify
drug resistance mutations and directly examine
parasite fitness and epistatic interactions with
other loci

Drug resistance genes typically interfere with key metabolic processes and therefore carry large fitness costs. We expect compensatory changes to evolve that restore fitness to drug resistant parasites. Different genes involved in drug resistance evolution may interact together to determine both drug resistance and fitness of parasites – these may act in additive and epistatic manner. We are using several different approaches to identify and understand fitness costs and compensatory evolution in malaria, and to determine whether genes interact additively or epistatically to determine outcome phenotypes. In particular, we are using experimental evolution, longitudinal population genomics surveys and CRISPR/Cas9 approaches to better understand the role of fitness, compensation and epistasis in drug resistance evolution.

Representative Papers

  • Nair, Shalini, Xue Li, Grace A Arya, Marina McDew-White, Marco Ferrari, and Tim Anderson. 2022. “Nutrient Limitation Magnifies Fitness Costs of Antimalarial Drug Resistance Mutations”. Antimicrobial Agents and Chemotherapy 66 (5): e0152921. https://doi.org/10.1128/aac.01529-21.

    Drug resistance mutations tend to disrupt key physiological processes and frequently carry fitness costs, which are a central determinant of the rate of spread of these mutations in natural populations. Head-to-head competition assays provide a standard approach to measuring fitness for malaria parasites. These assays typically use a standardized culture medium containing RPMI 1640, which has a 1.4- to 5.5-fold higher concentration of amino acids than human blood. In this rich medium, we predict that fitness costs will be underestimated because resource competition is weak. We tested this prediction using an artemisinin-sensitive parasite edited to contain kelch-C580Y or R561H mutations conferring resistance to artemisinin or synonymous control mutations. We examined the impact of these single amino acid mutations on fitness, using replicated head-to-head competition experiments conducted in media containing (i) normal RPMI, (ii) modified RPMI with reduced amino acid concentration, (iii) RPMI containing only isoleucine, or (iv) 3-fold diluted RPMI. We found a significant 1.3- to 1.4-fold increase in fitness costs measured in modified and isoleucine-only media relative to normal media, while fitness costs were 2.5-fold higher in diluted media. We conclude that fitness costs are strongly affected by media composition and will be significantly underestimated in normal RPMI. Several components differed between media, including pABA and sodium bicarbonate concentrations, so we cannot directly determine which is responsible. Elevated fitness costs in nature will limit spread of artemisinin (ART) resistance but will also promote evolution of compensatory mutations that restore fitness and can be exploited to maximize selection in laboratory experiments.

  • Nair, Shalini, Xue Li, Grace A Arya, Marina McDew-White, Marco Ferrari, Francois Nosten, and Tim J C Anderson. (2018) 2018. “Fitness Costs and the Rapid Spread of Kelch13-C580Y Substitutions Conferring Artemisinin Resistance.”. Antimicrobial Agents and Chemotherapy 62 (9). https://doi.org/10.1128/AAC.00605-18.

    Fitness costs are key determinants of whether drug resistance alleles establish and how fast they spread within populations. More than 125 different kelch13 alleles, each containing a different amino acid substitution, have arisen in Southeast Asian malaria parasite (Plasmodium falciparum) populations under artemisinin selection over the past 15 years in a dramatic example of a soft selective event. However, just one of these alleles (C580Y) is now outcompeting other alleles in multiple different countries and is spreading toward fixation. Here we examine the fitness consequences of C580Y, relative to another less successful kelch13 mutation (R561H), to try to explain the distinctive dynamics of C580Y. We hypothesized that C580Y will show lower fitness costs than other kelch13 substitutions in the absence of artemisinin treatment. We used CRISPR/Cas9 methods to introduce single mutations (C580Y or R561H) or synonymous control edits into a wild-type parasite isolated on the Thailand-Myanmar border, conducted replicated head-to-head competition assays, and determined the outcome of competition using deep sequencing of kelch13 amplicons. Contrary to our predictions, these experiments reveal that C580Y carries higher fitness costs (s [selection coefficient] = 0.15 ± 0.008 [1 standard error {SE}]) than R561H (s = 0.084 ± 0.005). Furthermore, R561H outcompetes C580Y in direct competition (s = 0.065 ± 0.004). We conclude that fitness costs of C580Y in isolation are unlikely to explain the rapid spread of this substitution.