Abstract
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.
Highlights
Fitness costs are key determinants of whether drug resistance alleles establish and how fast they spread within populations
This would allow parasites bearing C580Y to outcompete parasites carrying other kelch13 substitutions within mixed infections and to reach high parasite densities needed for transmission more rapidly than parasites carrying other alleles in monoclonal infections. We examine this hypothesis by introducing C580Y or another kelch13 substitution (R561H) onto the same wild-type genetic background and directly quantifying fitness costs during in vitro parasite culture using replicated head-to-head competition experiments
We used CRISPR/Cas9 to generate 4 edited parasite clones derived from NHP4302 (T1/2P ϭ 1.98 h), a cloned kelch13 wild-type parasite (Fig. 1A)
Summary
Fitness costs are key determinants of whether drug resistance alleles establish and how fast they spread within populations. More than 125 different kelch 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. We hypothesized that C580Y will show lower fitness costs than other kelch substitutions in the absence of artemisinin treatment. Resistance alleles typically disrupt parasite metabolic pathways and so are expected to carry a fitness cost in the absence of drug treatment [1, 2]. In Southeast Asia, at least 125 independent nonsynonymous substitutions in the kelch locus conferring artemisinin (ART) resistance (ART-R) have arisen in malaria (Plasmodium falciparum) populations treated with artemisinin-based combination therapies [5], in a dramatic example of a soft selective sweep. In the largest treatment failure study to date, there was no association between infections bearing ART-R kelch mutations and gametocyte carriage [14]
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